U.S. patent application number 12/089816 was filed with the patent office on 2008-11-20 for development of prodrugs possessing a nitric oxide donor diazen-1-ium-1,2-diolate noiety using in vitro/in silico predictions.
Invention is credited to Christopher Mark Diaper, Yi-Chan James Lin, Douglas Thacher Ridgway, Hugh A. Semple, Brian Duff Sloley, Yun Kau Tam.
Application Number | 20080288176 12/089816 |
Document ID | / |
Family ID | 40028387 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080288176 |
Kind Code |
A1 |
Tam; Yun Kau ; et
al. |
November 20, 2008 |
Development of Prodrugs Possessing a Nitric Oxide Donor
Diazen-1-Ium-1,2-Diolate Noiety Using in Vitro/in Silico
Predictions
Abstract
The present invention provides a method of using a
physiologically-based pharmacokinetic model to select a prodrug
molecule (NO--X) comprising a therapeutic agent X (e.g.
nonsteroidal anti-inflammatory drug, (NSAID)) and an appropriate
nitric oxide donor NO. The NSAID can be a non-selective or
selective cyclooxygenase inhibitor or other biocompatible compound
comprising a carboxyl group. The pharmacokinetic model uses in
vitro and/or in silico data to estimate an optimal set of
parameters that can predict whether a particular NO--X candidate is
capable of producing desirable therapeutic effects, e.g. enhanced
anti-inflammatory activity, reduced intestinal, cardiac and renal
toxicity. Accordingly, the present invention can greatly enhance
proper selection of an appropriate candidate for drug development,
thereby minimizing development time and conserving costs.
Inventors: |
Tam; Yun Kau; (Hong Kong,
CN) ; Diaper; Christopher Mark; (Edmonton, CA)
; Semple; Hugh A.; (Edmonton, CA) ; Ridgway;
Douglas Thacher; (Edmonton, CA) ; Lin; Yi-Chan
James; (Edmonton, CA) ; Sloley; Brian Duff;
(Edmonton, CA) |
Correspondence
Address: |
LAW OFFICES OF ALBERT WAI-KIT CHAN, PLLC
WORLD PLAZA, SUITE 604, 141-07 20TH AVENUE
WHITESTONE
NY
11357
US
|
Family ID: |
40028387 |
Appl. No.: |
12/089816 |
Filed: |
October 13, 2006 |
PCT Filed: |
October 13, 2006 |
PCT NO: |
PCT/US2006/040382 |
371 Date: |
June 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60726530 |
Oct 13, 2005 |
|
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|
Current U.S.
Class: |
702/19 ; 534/556;
534/557; 548/535; 562/568 |
Current CPC
Class: |
G16C 20/30 20190201;
C07D 207/16 20130101 |
Class at
Publication: |
702/19 ; 534/556;
548/535; 562/568; 534/557 |
International
Class: |
C07C 245/24 20060101
C07C245/24; C07D 207/16 20060101 C07D207/16; C07C 215/12 20060101
C07C215/12; G06F 19/00 20060101 G06F019/00 |
Claims
1-35. (canceled)
36. A method of pairing a therapeutic agent with an appropriate
nitric oxide donor to create an effective prodrug molecule,
comprising: (i) obtaining in vitro or in silico pharmacokinetic
and/or pharmacodynamic data of the therapeutic agent, nitric oxide
donor and nitric oxide from said donor; (ii) placing the data into
a physiologically-based pharmacokinetic/pharmacodynamic model
comprising: (a) a first compartment model which divides a
gastrointestinal tract into compartments, wherein said compartment
model describes gastrointestinal absorption of said prodrug
molecule; and (b) a second compartment model which divides a body
into plasma/blood and tissue compartments, wherein said compartment
model describes the time course of the therapeutic agent, the
nitric oxide donor, and nitric oxide in gastrointestinal tract,
blood, and tissues; and (iii) generating output parameters from
said pharmacokinetic model, wherein said output parameters
determine the appropriateness of pairing of said therapeutic agent
with said nitric oxide donor to create an effective prodrug
molecule.
37. The method of claim 1, wherein the first compartment model
divides a gastrointestinal tract into seven compartments, wherein
the tissue compartments are selected from the group consisting of
heart, liver, and kidney, and wherein the therapeutic agent is
selected from the group consisting of nonsteroidal
anti-inflammatory drugs and antibiotics.
38. The method of claim 1, wherein the in vitro or in silico data
are selected from the group consisting of pKa values, octanol/water
partition coefficients, solubility data, log P values, permeability
values, metabolism data, hydrolysis data, serum protein binding
data, nitric oxide release rate, pharmacokinetic and
pharmacodynamic data of a prodrug and a therapeutic agent, and
stability data in gastric and intestinal environments.
39. The method of claim 1, wherein the in vitro or in silico data
comprise volumes of distribution for the prodrug and the nitric
oxide donor.
40. A prodrug molecule selected by the method of claim 1, wherein
said prodrug molecule can be enzymatically or hydrolytically
cleaved to give a therapeutic agent and a nitric oxide donor.
41. The prodrug molecule of claim 5, wherein the therapeutic agent
is a nonsteroidal anti-inflammatory drug or an antibiotic.
42. The prodrug molecule of claim 5, wherein the nitric oxide donor
is diazen-1-ium-1,2-diolate.
43. The prodrug molecule of claim 6, wherein the nonsteroidal
anti-inflammatory drug is a non-selective cyclooxygenase isozyme
inhibitor or a cyclooxygenase-2 inhibitor.
44. The prodrug molecule of claim 8, wherein the non-selective
cyclooxygenase isozyme inhibitor is selected from the group
consisting of acetylsalicylic acid (CH.sub.3COOC.sub.6H.sub.4COOH),
IBUPROFEN (C.sub.13H.sub.18O.sub.2), NAPROXEN
(C.sub.14H.sub.14O.sub.3,), indomethacin
(C.sub.19H.sub.16ClNO.sub.4), and diclofenac
(Cl.sub.14H.sub.10Cl.sub.2NNaO); or the cyclooxygenase-2 inhibitor
comprises a carboxyl group.
45. The prodrug molecule of claim 5, wherein said donor has a
half-life that is longer than the total time period for hydrolysis
and absorption, wherein a therapeutic dosage of nitric oxide is
released into enterocytes, thereby protecting them against damage
caused by gastrointestinal irritation, bleeding or ulceration, or
wherein a therapeutic dosage of nitric oxide is released into the
blood stream, thereby protecting one or more organ systems, wherein
the organ systems are selected from the group consisting of heart,
kidney, and cardiovascular system.
46. The prodrug molecule of claim 5 comprising: (i) a nitric oxide
donor linked to an amino acid through a linkage that is susceptible
to enzymatic hydrolysis or cleavage; and (ii) a therapeutic agent
directly linked to said amino acid, or linked to said amino acid
through a spacer, wherein the linkage between the therapeutic agent
and the spacer, or the linkage between the spacer and the amino
acid is susceptible to enzymatic hydrolysis or cleavage, wherein
release of the nitric oxide donor and the therapeutic agent from
the prodrug molecule can be controlled independently.
47. The prodrug molecule of claim 11, wherein the amino acid is
selected from the group consisting of hydroxyproline, glutamic
acid, and aspartic acid, and wherein the linkage susceptible to
enzymatic hydrolysis or cleavage is selected from the group
consisting of ester linkage, thioester linkage, amide linkage, and
sulfonamide linkage.
48. The prodrug molecule of claim 11, wherein said therapeutic
agent is a nonsteroidal anti-inflammatory drug or an antibiotic,
and said donor is diazen-1-ium-1,2-diolate.
49. The prodrug molecule of claim 5 with the formula of:
##STR00034## wherein R.sup.1 is the uncarboxylated core of a
non-steroidal anti-inflammatory drug, or a structure of formula II:
##STR00035## wherein R.sup.8 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, or an unsubstituted or
substituted C.sub.3-12 branched chain alkyl; wherein X.sup.1 has a
formula selected from the group consisting of: (i) formula III:
##STR00036## wherein X.sup.2 is oxygen, sulfur, or NH, and X.sup.3
is oxygen, sulfur, or NH, (ii) formula IV: ##STR00037## wherein
X.sup.4 is oxygen, sulfur, or NH, and X.sup.5 is oxygen, sulfur, or
NH, (iii) formula V: ##STR00038## and (iv) formula VI: ##STR00039##
wherein X.sup.6 is oxygen, sulfur, or NH; wherein R.sup.2 is
hydrogen, an unsubstituted or substituted C.sub.1-12 straight chain
alkyl, or an unsubstituted or substituted C.sub.3-12 branched chain
alkyl; wherein R.sup.3 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, or an unsubstituted or substituted
C.sub.3-12 branched chain alkyl; wherein R.sup.4 is selected from
the group consisting of: (i) hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, or an
unsubstituted or substituted heteroaryl, (ii) formula VII:
##STR00040## wherein R.sup.9 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, an amide derivative linked via a carboxy
group of an amino acid, or an amide derivative of a polypeptide,
(iii) formula VIII: ##STR00041## wherein X.sup.7 is oxygen, sulfur,
or NH, and R.sup.10 is an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, and (iv) formula IX: ##STR00042## wherein
X.sup.8 is oxygen, sulfur, or NH; and R.sup.11 is a hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl; and R.sup.12
is a hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl; wherein R.sup.5 is hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, an unsubstituted or substituted heteroaryl, a structure of
formula VII, a structure of formula VIII, or a structure of formula
IX; wherein R.sup.6 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, a structure of formula VII, or a structure of formula
VIII; wherein R.sup.7 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, a structure of formula VII, or a structure of formula
VIII, or a structure of formula XI: ##STR00043## wherein X.sup.9 is
oxygen, sulfur, or NH, and R.sup.14 is hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, or an amino acid wherein X.sup.9 is the
amino group of the amino acid; and wherein Y is a structure of the
formula XIII: ##STR00044## or a structure of the formula XIV:
##STR00045## wherein R.sup.15 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl.
50. The prodrug molecule of claim 14, wherein NR.sup.6R.sup.7 is a
heterocycle of (i) formula X: ##STR00046## wherein R.sup.13 is
hydrogen, or (ii) formula XII: ##STR00047## wherein R.sup.4 is
selected from the group consisting of: (i) hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl, (ii) formula
VII: ##STR00048## wherein R.sup.9 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, an amide derivative linked via a carboxy
group of an amino acid, or an amide derivative of a polypeptide,
(iii) formula VIII: ##STR00049## wherein X.sup.7 is oxygen, sulfur,
or NH, and R.sup.10 is an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, and (iv) formula IX: ##STR00050## wherein
X.sup.8 is oxygen, sulfur, or NH; and R.sup.11 is a hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl; and R.sup.12
is a hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl.
51. The prodrug molecule of claim 5 with the formula of
##STR00051## wherein Z is a structure of the formula XIII,
##STR00052## or a structure of the formula XIV, ##STR00053##
wherein R.sup.15 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl; wherein R.sup.1 is the uncarboxylated core of a
non-steroidal anti-inflammatory drug, or a structure of formula II:
##STR00054## wherein R.sup.8 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, or an unsubstituted or
substituted C.sub.3-12 branched chain alkyl; wherein R.sup.4 is
selected from the group consisting of: (i) hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl, (ii) formula
VII: ##STR00055## wherein R.sup.9 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, an amide derivative linked via a carboxy
group of an amino acid, or an amide derivative of a polypeptide,
(iii) formula VIII: ##STR00056## wherein X.sup.7 is oxygen, sulfur,
or NH, and R.sup.10 is an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, and (iv) formula IX: ##STR00057## wherein
X.sup.8 is oxygen, sulfur, or NH; and R.sup.11 is a hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl; and R.sup.12
is a hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl; wherein R.sup.5 is hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, an unsubstituted or substituted heteroaryl, a structure of
formula VII, a structure of formula VIII, or a structure of formula
IX; wherein R.sup.6 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, a structure of formula VII, or a structure of formula
VIII; wherein R.sup.7 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, a structure of formula VII, or a structure of formula
VIII, or a structure of formula XI: ##STR00058## wherein X.sup.9 is
oxygen, sulfur, or NH, and R.sup.14 is hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, or an amino acid wherein X.sup.9 is the
amino group of the amino acid; and wherein Y is a structure of the
formula XIII: ##STR00059## or a structure of the formula XIV:
##STR00060## wherein R.sup.15 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl (an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl.
52. The prodrug molecule of claim 5 with the formula of
##STR00061## wherein R.sup.1 is the uncarboxylated core of a
non-steroidal anti-inflammatory drug, or a structure of formula II:
##STR00062## wherein R.sup.8 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, or an unsubstituted or
substituted C.sub.3-12 branched chain alkyl; wherein R.sup.4 is
selected from the group consisting of: (i) hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl, (ii) formula
VII: ##STR00063## wherein R.sup.9 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, an amide derivative linked via a carboxy
group of an amino acid, or an amide derivative of a polypeptide,
(iii) formula VIII: ##STR00064## wherein X.sup.7 is oxygen, sulfur,
or NH, and R.sup.10 is an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, and (iv) formula IX: ##STR00065## wherein
X.sup.8 is oxygen, sulfur, or NH; and R.sup.11 is a hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl; and R.sup.12
is a hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl; wherein R.sup.6 is hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, an unsubstituted or substituted heteroaryl, a structure of
formula VII, or a structure of formula VIII; wherein R.sup.7 is
hydrogen, an unsubstituted or substituted C.sub.1-12 straight chain
alkyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkyl, an unsubstituted or substituted C.sub.1-12 straight chain
alkenyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkenyl, an unsubstituted or substituted benzyl, an unsubstituted
or substituted phenyl, an unsubstituted or substituted C.sub.1-4
aryl alkyl, an unsubstituted or substituted heteroaryl, a structure
of formula VII, or a structure of formula VIII, or a structure of
formula XI: ##STR00066## wherein X.sup.9 is oxygen, sulfur, or NH,
and R.sup.14 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, or an amino acid wherein X.sup.9 is the amino group of
the amino acid; and wherein Y is a structure of the formula XIII:
##STR00067## or a structure of the formula XIV: ##STR00068##
wherein R.sup.15 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl.
53. The prodrug molecule of claim 5 with the formula of
##STR00069## wherein R.sup.18 is selected from the group consisting
of: (i) formula XVIII: ##STR00070## (ii) formula XIX: ##STR00071##
(iii) formula XX: ##STR00072## and (iv) formula XXI: ##STR00073##
wherein the substructure: ##STR00074## represents the core
structure of an amino acid selected from alanine, 2-aminobutyric
acid, acid, .alpha.-aminosuberic acid, arginine, asparagines,
aspartic acid, citrulline, .beta.-cyclohexylalanine, cysteine,
3,4-dehydroproline, glutamic acid, glutamine, glycine, histadine,
homocitrulline, homoserine, hydroxyproline, .beta.-hydroxyvaline,
isoleucine, leucine, lysine, methionine, norleucine, novaline,
ornithine, penicillamine, phenylalanine, phenylglycine, proline,
pyroglutamine, sarcosine, serine, threonine, tryptophan, tyrosine
and valine; wherein R.sup.1 is the uncarboxylated core of a
non-steroidal anti-inflammatory drug, or a structure of formula II:
##STR00075## wherein R.sup.8 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, or an unsubstituted or
substituted C.sub.3-12 branched chain alkyl; wherein R.sup.6 is
hydrogen, an unsubstituted or substituted C.sub.1-12 straight chain
alkyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkyl, an unsubstituted or substituted C.sub.1-12 straight chain
alkenyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkenyl, an unsubstituted or substituted benzyl, an unsubstituted
or substituted phenyl, an unsubstituted or substituted C.sub.1-4
aryl alkyl, an unsubstituted or substituted heteroaryl, a structure
of formula VII, or a structure of formula VIII; wherein R.sup.7 is
hydrogen, an unsubstituted or substituted C.sub.1-12 straight chain
alkyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkyl, an unsubstituted or substituted C.sub.1-12 straight chain
alkenyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkenyl, an unsubstituted or substituted benzyl, an unsubstituted
or substituted phenyl, an unsubstituted or substituted C.sub.1-4
aryl alkyl, an unsubstituted or substituted heteroaryl, a structure
of formula VII, or a structure of formula VIII, or a structure of
formula XI: ##STR00076## wherein X.sup.9 is oxygen, sulfur, or NH,
and R.sup.14 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, or an amino acid wherein X.sup.9 is the amino group of
the amino acid; and wherein Y is a structure of the formula XIII:
##STR00077## or a structure of the formula XIV: ##STR00078##
wherein R.sup.15 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl.
54. The prodrug molecule of claim 5 with the formula of
##STR00079## wherein R.sup.4 is selected from the group consisting
of: (i) hydrogen, an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, (ii) formula VII: ##STR00080## wherein
R.sup.9 is hydrogen, an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, an amide derivative linked via a carboxy group of an
amino acid, or an amide derivative of a polypeptide, (iii) formula
VIII: ##STR00081## wherein X.sup.7 is oxygen, sulfur, or NH, and
R.sup.10 is an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, and (iv) formula IX: ##STR00082## wherein
X.sup.8 is oxygen, sulfur, or NH; and R.sup.11 is a hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl; and R.sup.12
is a hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl; wherein R.sup.5 is hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, an unsubstituted or substituted heteroaryl, a structure of
formula VII, a structure of formula VIII, or a structure of formula
IX; wherein R.sup.6 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, a structure of formula VII, or a structure of formula
VIII; and wherein R.sup.7 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, a structure of formula VII, or a
structure of formula VIII, or a structure of formula XI:
##STR00083## wherein X.sup.9 is oxygen, sulfur, or NH, and R.sup.14
is hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, or an amino acid wherein X.sup.9 is the amino group of
the amino acid.
55. The prodrug molecule of claim 5 with the formula of
##STR00084## wherein R.sup.4 is selected from the group consisting
of: (i) hydrogen, an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, (ii) formula VII: ##STR00085## wherein
R.sup.9 is hydrogen, an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, an amide derivative linked via a carboxy group of an
amino acid, or an amide derivative of a polypeptide, (iii) formula
VIII: ##STR00086## wherein X.sup.7 is oxygen, sulfur, or NH, and
R.sup.10 is an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl, and (iv) formula IX: ##STR00087## wherein
X.sup.8 is oxygen, sulfur, or NH; and R.sup.11 is a hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, or an unsubstituted or substituted heteroaryl; and R.sup.12
is a hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, or an unsubstituted or
substituted heteroaryl; wherein R.sup.5 is hydrogen, an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, an unsubstituted or substituted heteroaryl, a structure of
formula VII, a structure of formula VIII, or a structure of formula
IX; wherein R.sup.6 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, a structure of formula VII, or a structure of formula
VIII; and wherein R.sup.7 is hydrogen, an unsubstituted or
substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, a structure of formula VII, or a
structure of formula VIII, or a structure of formula XI:
##STR00088## wherein X.sup.9 is oxygen, sulfur, or NH, and R.sup.14
is hydrogen, an unsubstituted or substituted C.sub.1-12 straight
chain alkyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkyl, an unsubstituted or substituted C.sub.1-12 straight
chain alkenyl, an unsubstituted or substituted C.sub.3-12 branched
chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl, or an amino acid wherein X.sup.9 is the amino group of
the amino acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. provisional
applications Nos. 60/726,530, filed Oct. 13, 2005, 60/730,120,
filed Oct. 21, 2005, 60/756,446, filed Jan. 5, 2006, and
60/812,230, filed Jun. 9, 2006. The disclosure of the preceding
applications are hereby incorporated in their entireties by
reference into this application.
FIELD OF THE INVENTION
[0002] This invention relates to development of prodrug molecules
comprising a therapeutic agent (such as nonsteroidal
anti-inflammatory drug) and a nitric oxide donor.
BACKGROUND OF THE INVENTION
[0003] Since the introduction of aspirin over a hundred years ago,
nonsteroidal anti-inflammatory drugs (NSAIDs) have been used to
treat patients who suffer from various forms of arthritis. The
anti-inflammatory effects of NSAIDs are mainly mediated via the
inhibition of cyclooxygenase (COX)-derived prostaglandin (PG)
synthesis (Brooks et al., 1991; Cathella-Lawson et al., 2001;
Rodriguez et al., 2001; Vane et al., 1998). PG inhibition is also a
major side effect of NSAIDs, gastric ulcers (Wallace 2003). The
reason is that PG is responsible for vascular homeostasis and
gastrointestinal tract protection (Hollander, 1994; Rainsford,
1999; Schoen & Vender, 1989).
[0004] In the early 1990s, it was discovered that COX enzyme exists
in two isoforms, COX-1 and COX-2. Initially, COX-1 was thought to
be a constitutive and a ubiquitous enzyme that is present in a
number of tissues including the GI tract (Lipsky, 1999; Buttar and
Wang, 2000), whereas COX-2 was regarded as strictly an inducible
enzyme. Pro-inflammatory mediators such as cytokines, growth
factors, lipopolysaccharides or prostanoids would up-regulate this
isoform (Hinz et al., 2000; Hinz and Brune, 2002). In theory, an
inhibition of COX-2 isozyme would produce anti-inflammatory effects
while sparing the GI from damages. This discovery has led to the
development of selective COX-2 inhibitors. At the end of 1999, the
first COX-2 inhibitor, CELECOXIB, was introduced to the market.
Shortly after the introduction of CELECOXIB, a more specific COX-2
inhibitor, ROFECOXIB, was launched. Clinical studies showed that
these COX-2 inhibitors are as effective in fighting pain and
inflammation as the other non-selective NSAIDs (Morrison et al.,
1999; Reicin et al., 2001; Cannon et al., 2000; Day et al., 2000).
In a clinical trial, VIGOR (Bresalier et al., 2005), involving
8,076 patients suffering from rheumatoid arthritis, it was found
that ROFECOXIB has significantly less GI side effects when compared
to NAPROXEN. In another large clinical trial, CLASS (Silverstein et
al., 2000), the superiority of COX-2 inhibitor over non-selective
NSAID in the area of GI side effects was demonstrated by comparing
CELECOXIB with a non-selective NSAID, DICLOFENAC. The perceived
edge of COX-2 inhibitors over non-selective NSAIDs has led to its
huge success in the market place. In 2003, CELECOXIB and ROFECOXIB
took 75% of the US NSAID market (FitzGerald, 2003). Since then,
there has been a race to develop more selective COX-2 inhibitors
with better pharmacokinetic characteristics. Examples are
VALDECOXIB (or its prodrug PARECOXIB for parenteral use),
ETORICOXIB and LUMIRACOXIB.
[0005] In a clinical trial, APPROVe, conducted by Merck,
investigating the effects of ROFECOXIB in preventing colorectal
adenoma, it was discovered that ROFECOXIB, a selective COX-2
inhibitor, was associated with cardiovascular events such as
myocardial infarction and cerebral ischemia after 18 months of use.
The results of this clinical trial were recently published in the
New England Journal of Medicine (Bresalier et al., 2005). The
discovery of these potential side effects of ROFECOXIB has led to
its withdrawal from the market. Subsequently, the other two COX-2
inhibitors have come under close scrutiny. Basically, it is
questioned whether the cardiovascular events are limited to
ROFECOXIB or is it a class effect. On Apr. 7, 2005, the Food and
Drug Administration (FDA) requested that Pfizer suspend sales of
BEXTRA (VALDECOXIB) in the United States. FDA now requires all
prescription NSAIDs to provide additional information concerning
cardiovascular and gastrointestinal risks.
[0006] In order to appreciate FDA's decision on warning labels for
all selective and non-selective COX inhibitors, an understanding of
arachidonic acid metabolism is necessary (see the following
diagram: an arachidonic acid cascade. CYP: cytochrome P.sub.450
isozymes; EETs: epoxyeicosatrienoic acids; HETEs:
hydroxyeicosatetraenoic acids; HODEs: hydroxyoctadecadienoic acids;
DP: prostaglandin D.sub.2 receptor; EP: prostaglandin E.sub.2
receptor; IP: prostacyclin receptor; FP: prostaglandin F.sub.2
receptor; TP: prostaglandin T.sub.2 receptor.)
##STR00001##
[0007] Arachidonic acid (AA) is a metabolic product of
membrane-bound phospholipids through phospholipase A.sub.2. AA is a
substrate of cyclooxygenase and peroxidase to generate an
endoperoxide, prostaglandin H.sub.2 (PGH.sub.2) (Warner and
Mitchell, 2004; Davidge 2001; FitzGerald 2003a). PGH.sub.2 is the
precursor of thromboxane A.sub.2 (TxA.sub.2), prostanoids and
prostacyclins. These reactions are mediated through tissue specific
enzymes thromboxane synthase, prostanoid synthase and prostacyclin
synthase, respectively. Inhibition of COX-1 isoform will lead to a
reduction in the circulatory thromboxane and prostaglandin levels.
In addition to other tissues, COX-1 is found in the platelet and
the GI tract. Since platelet aggregation is atherogenic, a
reduction of thromboxane in blood would reduce the risk of thrombi
formation and therefore cardiovascular ischemia (Krotz et al.,
2005). On the other hand, inhibition of COX-2 isozyme will lead to
a reduction of PGI.sub.2. This prostacyclin is a potent vasodilator
and an anti-platelet agent (Krotz et al., 2005). Inhibition of
COX-2 isozyme has been postulated to cause the untoward
cardiovascular events that have been observed in patients (Krotz et
al., 2005). Non-selective COX inhibitors such as the traditional
NSAIDs (e.g. ASPIRIN, IBUPROFEN, NAPROXEN, INDOMETHACIN, etc.) have
various degrees of COX-1 and 2 inhibition; therefore, they may have
various degree of cardiovascular risks.
[0008] In a review written by Krotz et al. (2005), it was
postulated that the balance between thromboxane and PGI.sub.2 is a
crucial determinant of cardiovascular events for both selective and
non-selective NSAIDs. This has led to the postulation that the
selectivity of COX inhibition has an important role to play in the
cardiovascular safety of NSAIDs. Besides the ratio of COX-1/COX-2
selectivity, pharmacokinetic properties, dosage and the
cardiovascular state of patients are also factors that can
contribute to the observed cardiovascular risks.
[0009] Contrary to previous beliefs, the COX-2 isozyme is not only
inducible, it is also expressed constitutively (Zimmermann et al.,
1998; Iseki 1995; Nantel et al., 1999; Chakraborty et al., 1996;
Slater et al., 1999, 1999a; Damm et al., 2001; Tegeder et al.,
2000). This enzyme is expressed in various tissues including gut
(Zimmermann et al., 1998; Iseki 1995), myometrium (Slater et al.,
1999, 1999a) and kidneys (Tegeder et al., 2000). COX-2 inhibition
can lead to sodium and fluid retention which may lead to
hypertension (Krotz, 2005). Hypertension is a known atherogenic
factor.
[0010] Nitric oxide (NO) is now widely recognized as a critical
mediator of gastrointestinal mucosal defense, exerting many of the
same actions as prostaglandins in the gastrointestinal tract
(Wallace 2003). NO has been shown to reduce the severity of gastric
injury in experimental models (McNaughton et al., 1989; Kitagawa et
al., 1990). It has been proposed that linking a NO-releasing moiety
to a NSAID may reduce the toxicity of the latter (Wallace et al.
1994). In animal studies, NO-releasing derivatives of a wide range
of NSAIDs (FIG. 1), including NO-aspirin, NO-naproxen,
NO-flurbiprofen, and NO-diclofenac, have been shown to spare the
gastrointestinal tract, even though they suppressed prostaglandin
synthesis as effectively as the parent drug (Wallace et al., 1994a
and 1994b; Reuter et al., 1994; Cuzzolin et al., 1995; Davies
1997). A number of other NO-NSAIDS have been disclosed which
utilize nitrooxyalkyl functionality as the source of NO (Ranatunge
et al., 2006; Kartasasmita et al., 2002; Andersson et al., 2004;
Gilmer et al., 2002; Almirante et al., 2006; Benedini et al., 2000;
Bolla et al., 2005; Rivolta et al., 2005; Del Soldato 2002a, 2002b,
2003, 2004a and 2004b; Bandarage et al., 2000; Earl et al., 2004;
Satyam 2006).
[0011] However, an important drawback to this design is that
production of NO from organic nitrate esters has been reported to
occur via a number of mechanisms, both enzymatic (glutathione
S-transferase, cytochrome P-450 and other uncharacterized enzymes)
and chemical (non-proteinous thiols) (Fung 2004). Organic nitrate
esters also demonstrate a reduction of efficiency on continued use
of the drugs, contributing to "nitrate tolerance" (Const and
Ferdinandy 2005). In this regard, N-diazen-1-ium-1,2-diolates (also
referred to as diazeniumdiolates or NONOates) have the potential to
release up to 2 equivalents of NO with half-lives that correlate
well with their pharmacological durations of action. These
observations suggest that O.sup.2-unsubstituted diazeniumdiolates
are minimally affected by metabolism, and are essentially different
from currently available clinical vasodilators (Keefer 2003).
[0012] O.sup.2-Substituted diazeniumdiolates possess three
attributes that make them especially attractive for designing drugs
to treat a variety of disease states, namely structural diversity,
dependable rates of NO release, and rich derivatization chemistry
that facilitates targeting of NO to specific target organ and/or
tissue sites (Keefer 2003). Unsubstituted diazeniumdiolates may be
derivatized at the O.sup.2 position to form NO donors which are
much more resistant to physiological conditions, resulting in a
pronounced increase in the half life of the NO donor. Saavedra et
al. (1999) reported a NO-NSAID based on an O.sup.2-methoxy
substituted diazeniumdiolate derived from piperazine. The NSAID
IBUPROFEN was covalently attached to the distal nitrogen of the
piperazine linker via an amide bond. The O.sup.2-methoxy
diazeniumdiolate spontaneously released NO under physiological
conditions with a half life of approximately 17 days.
Alternatively, diazeniumdiolates can be substituted at the
O.sup.2-position with acetyloxy methyl functionality which is
resistant to physiological conditions but susceptible towards
enzymatic hydrolysis on exposure to esterases (Saavedra et al.
2000). These NO generating moieties can be linked to other
biocompatible compounds such as NSAIDS so that the NSAID and
unsubstituted diazeniumdiolate are enzymatically released. Knaus et
al. (2005) disclosed a series of novel NSAID molecules of this type
possessing diazeniumdiolates as NO donors. These molecules have
been shown to have excellent gastric protective effects in rats.
However, the profiles of NO-NSAID and its active metabolites, NO
donor and NSAID, absorption and disposition have not been
elucidated. Furthermore, the effects of these candidates on kidney
and cardiovascular function are not known.
[0013] In light of the COX-2 inhibitor debacle, there is an
interest in developing an NO-NSAID using COX-2 inhibitors. It has
been shown that NO has beneficial effects on the cardiovascular
system and the kidneys (Mollace et al., 2005). It would be logical
to synthesize and/or to administer a COX-2 inhibitor with a NO
donor. Connor and Manning (2005) described a method comprising the
administration of a combination of a COX-2 inhibitor and a nitric
oxide donating agent.
[0014] Unlike traditional non-selective COX inhibitors which
possess a carboxyl group for forming a covalent linkage with a
nitric oxide donor such as diazeniumdiolates, COX-2 inhibitors like
ROFECOXIB do not always have a functional handle that would readily
allow the attachment of a nitric oxide donor moiety. However, it
has been reported that certain COX-2 inhibitors, and prodrugs of
specific COX-2 inhibitors (for example ROFECOXIB) do contain
carboxylic acids that can be covalently bound to a nitrooxyalkyl NO
donor via an ester linkage (Engelhardt et al., 2006; Del Soldato et
al., 2004c; Letts et al., 2003). Some COX-2 inhibitors such as
CELECOXIB and VALDECOXIB contain sulfonamide functionality that has
been used as a site of covalent linkage to a nitrooxyalkyl NO donor
(Del Soldato et al., 2004c; Bandarage et al., 2004). Alternate
strategies for attaching NO donors to COX-2 inhibitors include
pyrazoles containing a nitrate ester (ONO.sub.2) moiety as a nitric
oxide (NO)-donor (Ranatunge et al., 2004; Khanapure et al., 2002).
Bandarage et al. (2003) formed nitrosated and nitrosylated COX-2
inhibitors through one or more sites such as oxygen (hydroxyl
condensation), sulfur (sulfhydryl condensation) and/or nitrogen.
Dhawan et al. (2005) studied the pharmacology of a nitrated
VALDECOXIB derivative,
4-{5-[(nitrooxy)methyl]-3-phenylisoxazol-4-yl}benzenesulfonamide.
Khanapure et al. (2004) have synthesized a series of nitric oxide
derivatives of COX-2 inhibitors. A series of O.sup.2-unsubstituted
N-diazeniumdiolate salts was reported to be attached to the COX-2
inhibitor through an aryl nitrogen. The absence of substituent at
O.sup.2 suggests that the nitric oxide release from the
diazeniumdiolate derivative would follow that of other reported
O.sup.2-unsubstituted N-diazeniumdiolates. Therefore, it is assumed
that these derivatives release NO directly and do not require the
action of enzymes in vivo for this to occur. This type of
derivative may also lack tissue specificity in terms of NO donor
and NO release. The O.sup.2-substituted diazeniumdiolates
synthesized by Knaus et al. (2005), on the other hand, require the
action of esterases to release the NO donor and subsequently, NO.
Tissue specific delivery of NO, to some extent, can be accomplished
by adjusting the molecular structure to achieve a desired
hydrolysis rate in various organs such as the GI tract, liver,
blood, etc. However, the adjustment of the hydrolysis rate has not
been taken into consideration as the pharmacokinetics of these
moieties is unknown.
[0015] The NO-donating diazeniumdiolate NO-NSAIDs described by
Knaus, et al. (2005) are designed to be released in blood by serum
esterases. This approach of NO-NSAID design may not be optimal.
Esterases are traditionally known to be non-specific. However,
recent studies show that there are higher concentrations of certain
esterases in specific organs such as liver and intestine. It has
been shown that exposure of orally administered NO-NSAIDs, which
include the ones synthesized by Knaus et al. (2005), NCX-4016 and
AZD3582, to plasma is minimal. Hence, in the design of NO-NSAIDs, a
systematic approach which takes into account the drug-like
properties of these candidates is imperative. A flexible molecular
library of O.sup.2-substituted diazeniumdiolate NO-NSAID candidates
is required to generate and modify their properties in a controlled
fashion.
SUMMARY OF THE INVENTION
[0016] The present invention provides a physiologically based
pharmacokinetic/pharmacodynamic model. This model requires in
vitro/in silico input to estimate pharmacokinetic/pharmacodynamic
parameters of a test candidate. This model is useful for: (1)
screening a NO-NSAID candidate for its suitability of development,
and (2) providing information for synthesis of a new NO-NSAID (both
selective and non-selective) candidate that may have a better
chance of success in the development process.
[0017] The physiologically based pharmacokinetic/pharmacodynamic
model of the present invention contains a series of compartments
that describe the time course of a nonsteroidal anti-inflammatory
prodrug, its active metabolites and nitric oxide release in
intestine, liver, kidneys, blood/plasma and heart after prodrug
administration. The time course of the prodrug, its active
metabolites and nitric oxide release can be simulated using a
series of in vitro and in silico inputs. The stability of each
component in the gastrointestinal lumen is estimated using data
collected from artificial gastric and intestinal juice. Intestinal
metabolism is estimated using intestinal microsomes and absorption
rate is estimated using permeability data collected from a cell
monolayer such as Caco-2. Hepatic elimination is estimated using
liver microsomes and stability in plasma is calculated using
degradation of each component in the media. Plasma protein binding
can either be measured using a standardized in vitro method or it
can be estimated using an in silico method. The distribution of
each component in various parts of the body is estimated using an
in silico method. The rate of nitric oxide release is estimated
using an in vitro endothelial cell model. The time course of
prodrug, its active metabolites and nitric oxide can be simulated
in human and animal using this physiologically based
pharmacokinetic/pharmacodynamic model provided that the
corresponding in vitro and in silico data are used as inputs.
[0018] This model has been used successfully to predict the time
course of NO-NSAID prodrugs and NSAID after prodrug administration.
Advantages and deficiencies of existing NO-NSAIDs were identified.
Based on these results, a general structure of an NO-NSAID which
would provide an optimal delivery of nitric oxide to the gut, heart
and kidneys has been designed. This NO-NSAID molecule contains an
NSAID molecule which is connected to a nontoxic linker (e.g. an
amino acid) through an alkyl diester. A nitric oxide donor is
attached to the linker through an ester bond on the other end. The
nitric oxide releasing moiety is preferably a diazeniumdiolate
[0019] NSAID applicable in the present invention includes, but is
not limited to, non-selective COX inhibitors such as
acetylsalicylic acid (ASA, CH.sub.3COOC.sub.6H.sub.4COOH),
IBUPROFEN (C.sub.13H.sub.18O.sub.2), naproxen (NAP,
C.sub.14H.sub.14O.sub.3,), indomethacin
(C.sub.19H.sub.16ClNO.sub.4), or diclofenac
(C.sub.14H.sub.10Cl.sub.2NNaO); selective COX-2 inhibitors such as
CELECOXIB which contain a sulfonamide group or prodrugs of
ROFECOXIB which contains a carboxyl group.
[0020] In one embodiment, the present invention provides a method
of pairing a therapeutic agent with an appropriate nitric oxide
donor to create an effective prodrug molecule. The method
comprises: (i) obtaining in vitro or in silico pharmacokinetic or
pharmacodynamic data, (ii) placing the data into a
physiologically-based pharmacokinetic model comprising a
compartment model which divides a gastrointestinal tract into
compartments, and a second compartment model which divides a body
into plasma/blood and tissue compartments, and (iii) generating
output parameters from the pharmacokinetic model, wherein the
output parameters determine the pairing of a therapeutic agent with
an appropriate nitric oxide donor to create an effective prodrug
molecule.
[0021] The present invention also provides a prodrug molecule
selected by the above method, wherein the prodrug molecule
comprises a therapeutic agent and a nitric oxide donor.
[0022] In another embodiment, there is provided a prodrug molecule
comprising a nonsteroidal anti-inflammatory drug and a nitric oxide
releasing moiety, wherein the moiety has a half-life that is longer
than the total time period for hydrolysis and absorption, and
wherein a therapeutic dosage of nitric oxide is released into
enterocytes, thereby protecting against damage caused by
gastrointestinal irritation, bleeding or ulceration.
[0023] The present invention also encompasses uses of the prodrug
molecules identified by the method described herein to provide
therapeutic treatments. The present invention also provides a kit
comprising the prodrug molecules identified or described
herein.
[0024] The present invention also provides a prodrug molecule
comprising: (i) a nitric oxide releasing moiety linked to an amino
acid through a linkage that is susceptible to enzymatic hydrolysis
or cleavage, and (ii) a therapeutic agent directly linked to said
amino acid, or linked to said amino acid through a spacer, wherein
the linkage between the therapeutic agent and the spacer, or the
linkage between the spacer and the amino acid is susceptible to
enzymatic hydrolysis or cleavage, wherein the release of the nitric
oxide releasing moiety and the therapeutic agent from the prodrug
molecule can be controlled independently.
[0025] In another embodiment, there is provided a compound with the
formula of:
##STR00002##
[0026] In another embodiment, there is provided a compound with the
formula of:
##STR00003##
[0027] The present invention also provides a compound with the
formula of:
##STR00004##
[0028] The present invention also provides a compound with the
formula of:
##STR00005##
[0029] The present invention also provides a compound with the
formula of:
##STR00006##
[0030] The present invention also provides a compound with the
formula of:
##STR00007##
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows the chemical structures of some representative
NO-NSAIDS (organic nitrates).
[0032] FIG. 2 shows the structures of N3-108 and N3-112.
[0033] FIG. 3 shows hydrolysis of PYRO-NO-ASA (N3-108) in human
intestinal microsomes.
[0034] FIG. 4 shows hydrolysis of PYRO/NO-ASA (N3-108) in human
liver microsomes.
[0035] FIG. 5 shows hydrolysis of DMA/NO-ASA (N3-112) in human
intestinal microsomes.
[0036] FIG. 6 shows hydrolysis of DMA/NO-ASA (N3-112) in human
liver microsomes.
[0037] FIG. 7 shows ulcer index of NAPROXEN, its two
diazeniumdiolate prodrug compounds and their comparators.
[0038] FIG. 8 shows AUC.sub.0-6h (.mu.M-h) of NAPROXEN after dosing
with its two diazeniumdiolate prodrug compounds and AZD3582.
[0039] FIG. 9 shows serum nitrate concentrations for NAPROXEN, its
two diazeniumdiolate prodrug compounds and their comparators.
[0040] FIG. 10 shows cardiac tissue prostacyclin (PGI2) to
thromboxane A2 (TXB2) ratios for NAPROXEN, its two diazeniumdiolate
prodrug compounds and their comparators.
[0041] FIG. 11 shows ratios of urine alanine aminopeptidase to
creatinine concentration (AAP/Cr) for NAPROXEN, its two
diazeniumdiolate prodrug compounds and their comparators.
[0042] FIG. 12 shows urine N-acetylglucosaminidase to creatinine
concentration ratios (NAG/Cr) for NAPROXEN, its two
diazeniumdiolate prodrug compounds and their comparators.
[0043] FIG. 13 shows the layout of the entire
pharmacokinetic/pharmacodynamic model with simplified organ
modules.
[0044] FIG. 14 shows the layout of the intestinal segment
modules.
[0045] FIG. 15 is a detailed layout of intestinal segment 1 showing
input from the stomach compartment and blood flow divided between
enterocytes and intestinal tissue.
[0046] FIG. 16 is a detailed layout of intestinal segment 2 showing
blood flow divided between enterocytes and intestinal tissue.
[0047] FIG. 17 is a detailed layout of intestinal segment 3 showing
blood flow divided between enterocytes and intestinal tissue.
[0048] FIG. 18 is a detailed layout of intestinal segment 4 showing
blood flow divided between enterocytes and intestinal tissue.
[0049] FIG. 19 is a detailed layout of intestinal segment 5 showing
blood flow divided between enterocytes and intestinal tissue.
[0050] FIG. 20 is a detailed layout of intestinal segment 6 showing
blood flow divided between enterocytes and intestinal tissue.
[0051] FIG. 21 is a detailed layout of intestinal segment 7 showing
blood flow divided between enterocytes and intestinal tissue.
[0052] FIG. 22 is a detailed layout of gastric compartment where
dose is introduced.
[0053] FIG. 23 is a detailed layout of heart compartment.
[0054] FIG. 24 is a detailed layout of kidney compartment.
[0055] FIG. 25 is a detailed layout of liver compartment showing
its dual (portal and arterial) blood supply.
[0056] FIG. 26 is a detailed layout of plasma, arterial and venous
compartments.
[0057] FIG. 27 is a detailed layout of tissue compartment showing
bidirectional distribution from the capillary bed into the
interstitial fluid and hence to the intracellular space and
back.
[0058] FIG. 28 is a detailed layout of lung compartment.
[0059] FIG. 29 shows a comparison between literature (circles) and
simulated results from the pharmacokinetic/pharmacodynamic model
for NAPROXEN after administration of 3 mg/kg naproxen in rats. - is
a line generated using the model. The shaded area is a two-fold
variation of the estimated values. The circles are data obtained
from FIG. 1 of Runkel et al. (1972).
[0060] FIG. 30 shows a comparison between literature (circles) and
simulated results from the pharmacokinetic/pharmacodynamic model
for NAPROXEN after administration of 300 mg naproxen in human. - is
a line generated using the model. The shaded area is a two-fold
variation of the estimated values. The circles are data obtained
from FIG. 6 of Runkel et al. (1972).
[0061] FIG. 31 is a comparison between literature (circles) and
simulated results from the pharmacokinetic/pharmacodynamic model
for AZD 3582 and NAPROXEN after administration of 15 .mu.mol/kg AZD
3582 in rats. - is a line generated using the model. The shaded
area is a two-fold variation of the estimated values. The circles
are data obtained from FIG. 3 of the Fagerholm preclinical paper
(2005).
[0062] FIG. 32 shows a comparison between literature (circles) and
simulated results from the pharmacokinetic/pharmacodynamic model
for AZD 3582 and NAPROXEN after administration of 375 mg AZD 3582
in human. - is a line generated using the model. The shaded area is
a two-fold variation of the estimated values. The circles are data
obtained from FIG. 6 of the Fagerholm clinical paper (2005).
[0063] FIG. 33 shows a model estimation of distribution of AZD 3582
in a rat after a 50 mg/kg of AZD 3582 was administered orally.
[0064] FIG. 34 shows a model estimation of distribution of AZD 3582
in a 70 kg human after a 50 mg/kg of AZD 3582 was administered
orally.
[0065] FIG. 35 shows a model estimation of the distribution
PYRO/NO-NAP (N-119) in a rat after a 50 mg/kg of N-119 was
administered orally.
[0066] FIG. 36 shows the Diazenium Diolate candidates reported by
Knaus et al., 2005.
[0067] FIG. 37 shows a general structure of the new generation
NO--NSAID which can be adjusted to provide systemic delivery of
NO.
[0068] FIG. 38 shows a COX2-AA-NONOate Prodrugs based on
Sulfonamide COX-2 Inhibitors.
[0069] FIG. 39 shows hydrolysis of di-NAPROXEN prodrug
(NAP-AA-NAP).
[0070] FIG. 40 shows the structure of DMA/NO-AA-DMA/NO.
[0071] FIG. 41 shows differential enzymatic hydrolysis of
NO-AA-NSAIDS.
[0072] FIG. 42 shows a pathway of hydrolysis for CMD113 and
CMD114.
[0073] FIG. 43 shows a second pathway of hydrolysis for CMD113 and
CMD114.
[0074] FIG. 44 shows hydrolysis of CMD113 in rat intestinal
microsomes. Naproxen-AA and NO-AA are meant to be trends only, they
are not quantitative.
[0075] FIG. 45 shows hydrolysis of CMD113 in rat liver microsomes.
Naproxen-AA and NO-AA are meant to be trends only, they are not
quantitative.
[0076] FIG. 46 shows hydrolysis of CMD113 in human intestinal
microsomes. Naproxen-AA and NO-AA are meant to be trends only, they
are not quantitative.
[0077] FIG. 47 shows hydrolysis of CMD113 in human liver
microsomes. Naproxen-AA and NO-AA are meant to be trends only, they
are not quantitative.
[0078] FIG. 48 shows hydrolysis of CMD114 in rat intestinal
microsomes. Naproxen-AA and NO-AA are meant to be trends only, they
are not quantitative.
[0079] FIG. 49 shows hydrolysis of CMD114 in rat liver microsomes.
Naproxen-AA and NO-AA are meant to be trends only, they are not
quantitative.
[0080] FIG. 50 shows hydrolysis of CMD114 in human intestinal
microsomes. Naproxen-AA and NO-AA are meant to be trends only, they
are not quantitative.
[0081] FIG. 51 shows hydrolysis of CMD114 in human liver
microsomes. Naproxen-AA and NO-AA are meant to be trends only, they
are not quantitative.
[0082] FIG. 52 shows a proposed differential hydrolysis of
NO-AA-COX2 prodrugs.
[0083] FIG. 53 shows a general method for the preparation of
nonoate-amino acid-NAPROXEN prodrugs 26.
[0084] FIG. 54 shows a general method for the preparation of N-Ac
NAP-Glu-NAP 9.
[0085] FIG. 55 shows a general method for the preparation of N-Ac
DMA/NO-Glu-NO/DMA 14.
[0086] FIG. 56 shows a general method for the preparation of CMD113
and CMD114.
DETAILED DESCRIPTION OF THE INVENTION
[0087] In order to produce a lead with a reasonable chance of
success in clinical trials, it is imperative to understand the
relative pharmacokinetic and pharmacodynamic of the NSAID, the
diazeniumdiolate derivative and the NO donor. This set of
information is important for selecting an appropriate NO donor for
a NSAID. The present invention provides a process by which a
physiologically based pharmacokinetic/pharmacodynamic model
requiring in vitro and in silico input is used to predict the
pharmacokinetic and pharmacodynamic behaviors of the prodrug
moiety.
[0088] The selection of in vitro tests is designed to provide
parameters for the above mentioned model. The use of an
inappropriate test would result in wrong predictions. For example,
Knaus et al. (2005) used guinea pig serum and porcine esterases to
hydrolyze their O.sup.2-substituted diazeniumdiolate derivatives of
ASPIRIN, as disclosed in U.S. Ser. No. 60/681,842. These tests
provided very little information in terms of in vivo human NO-NSAID
metabolism rate and the extent to which metabolic conversion to NO
donor and NSAID occurred. However, when some of these candidates
such as PYRO-NO-ASA (N3-108) and DMA-NO-ASA (N3-112) (FIG. 2) were
incubated with human intestinal and liver microsomes (FIGS. 3 to
6), it was found that they are rapidly hydrolyzed. The half-life
values were less than one minute. Based on these observations, it
has become apparent that prior to entering the systemic
circulation, it is likely that the NO-NSAID would have been
hydrolyzed in the enterocytes and little or no NO-NSAID would have
been detected in the blood stream. Furthermore, the release of NO
will commence immediately once the NO donor is detached from the
NSAID.
[0089] Prodrug design using esterases to release the active
principle has been commonly employed (Beaumont et al., 2003). The
function and distribution of esterases have been studied
extensively, particularly in the last few years. Although esterases
are known to be non-specific, there are dramatic inter-species and
inter-organ differences. The use of wrong esterases for prodrug
development has led to wrong lead selection and therefore, failure
(Beaumont et al., 2003; Mizen and Burton 1998).
[0090] The release of NO donor in enterocytes provides a higher
probability of gastrointestinal protection. However, it is not
certain whether NO-NSAID containing diazeniumdiolates would provide
protection to other organs such as the heart and kidneys. Gao's
group (Frehm et al., 2004) along with other research groups (Singel
et al., 2005) have been investigating hemoglobin as a nitric oxide
carrier in the blood. This is a hypothesis that describes systemic
delivery of nitric oxide to various tissues. In his latest
commentary, Gao (2005) stated, "Nitric oxide should never be
considered as a solitary and discrete chemical entity in any
biological systems."
[0091] Tissue specific delivery may be improved using an
appropriate diazeniumdiolate molecule. Keefer and his co-workers
have developed a series of diazeniumdiolates with NO generating
half-lives ranging from 2 seconds to over 20 hours (Keefer, 2005).
Furthermore, Keefer et al. has functionalized diazeniumdiolates
with carbohydrates so that the NO is released by the action of
glucosidases, thereby limiting NO release to tissues containing
this class of enzyme (Showalter et al., (2005). However, in the
absence of a systematic approach, the selection of an appropriate
diazeniumdiolate is challenging.
[0092] The challenge of developing a successful prodrug molecule
NO--X (e.g. NO-NSAID) for the treatment of arthritis,
cardiovascular and other ailments including cancer is due largely
to the difficulty of obtaining the desired rate, extent, and site
of nitric oxide release.
[0093] For example, if a molecule of a NO-NSAID is predominantly
absorbed into the systemic circulation after oral administration,
gastric and intestinal membranes can be protected from ulceration
only by the nitric oxide released in the blood. The concentration
of nitric oxide in the stomach and intestine may not be high enough
because NO donor concentration in the blood will be at least an
order of magnitude lower than the concentration existing locally in
the intestine during the absorption process.
[0094] If the NO donor has a short half-life, this will probably
not be sufficient to protect the gastrointestinal tract from a
NSAID with a longer half-life because the NO released has a very
short duration in the body.
[0095] Rapid release of NO donor from NO-NSAID in the enterocytes
during the absorption process may provide optimal gastrointestinal
protection; however, the concentration of nitric oxide in other
organs such as heart and kidneys may not be high enough for
protection because the NO never reaches the systemic
circulation.
[0096] The present invention provides a physiologically based
pharmacokinetic/pharmacodynamic model for estimating an optimal set
of parameters for chemically pairing an NSAID or other therapeutic
or biocompatible agents with an appropriate NO donor such as
diazeniumdiolate.
[0097] The prodrug design approach described herein is not only
applicable to NO-NSAID. Other therapeutic or biocompatible agents
can be linked to a NO donor such as diazeniumdiolate to optimize
delivery and release in specific organs. The use of a biocompatible
principle for this purpose is a design for diazeniumdiolate as the
sole therapeutic agent.
[0098] The pharmacokinetic/pharmacodynamic model of the present
invention describes the time course of absorption, distribution,
metabolism, NO release (FIGS. 13-28), and COX inhibition in animals
and human. This pharmacokinetic/pharmacodynamic model comprises a
seven compartment model to describe gastrointestinal absorption and
a number of physiological compartment models to describe the time
course of individual species in the rest of the body including
relevant organs and tissue reservoirs. Pharmacodynamic compartments
describing the time course of NO release and COX inhibition are
attached to the appropriate pharmacokinetic compartments (FIG. 13).
The same pharmacokinetic/pharmacodynamic model can be easily
adapted to describe the time course of other prodrug moieties. In
one embodiment, input parameters of this model are obtained from a
series of in vitro tests or in silico estimates of the NO-NSAID or
its active and stable metabolite, for example, a molecule that
contains a diazeniumdiolate and a linker molecule:
[0099] Representative in vitro tests or in silico estimates
include: (a) pKa estimation or measurement; (b) Log P measurement
or in silico estimation; (c) Solubility in various physiological
fluids; (d) Permeability. Caco-2 and/or NOVOKIN's proprietary
animal and human cell lines can be used to obtain this parameter;
(e) Metabolic rate in the intestine and liver. Human or animal
intestinal microsomes, S9 fraction, and cytosol can be used for
this purpose. Human or animal hepatocytes, liver microsomes, S9
fraction, and cytosol can also be used; (f) Hydrolysis in human or
animal plasma; (g) Serum or plasma protein binding. It can be
measured in vitro or estimated in silico; (h) The rate of NO
release; (i) Existing pharmacokinetic and pharmacodynamic data of
NSAID or a biocompatible agent; (j) Existing NO release rate of
known diazeniumdiolate if applicable; (k) Stability in gastric and
intestinal environment; and (l) In silico volume of distribution
estimation.
[0100] Representative outputs of this simulation for a particular
NO--X (e.g. NO-NSAID) species are listed as follows: (a) Stability
of NO--X in the gastrointestinal tract; (b) Time course and extent
of NO--X absorption in the intestine; (c) Time course and extent of
NO and X release in the enterocytes; (d) Time course of NO
generation from the NO donor in various tissues including
gastrointestinal tract, liver, heart and kidneys; (e) Time course
of COX-1 inhibition in the intestine; (f) Time course of NO in
blood; (g) Time course of NO in tissues including gastrointestinal
tract, liver, heart and kidneys; (h) Time course of NO in blood and
tissues including gastrointestinal tract, liver, heart and kidneys;
(i) Estimation of systemic effect contributed by nitric oxide.
[0101] For example, an optimal candidate of NO-naproxen for
treating arthritis should have the following parameters: (a) Stable
under acidic and basic conditions; (b) Stable under
gastrointestinal environments; (c) Optimal hydrophilic/hydrophobic
properties; (d) Maximum absorption into enterocytes; (e)
Significant percentage of the NO-NSAID dose should be hydrolyzed
into NO donor and NSAID in the enterocytes; (f) NO donor should be
absorbed to a significant extent. Preferably, a significant
percentage of nitric oxide is released from the total NO donor into
enterocytes. The concentration of nitric oxide should be high
enough to protect the stomach and intestinal tract from irritation,
bleeding and ulceration. A significant percentage of the nitric
oxide donor should be released in the gastrointestinal tract,
preferably, 5 to 50% of the dose equivalent; (g) The NO donor
should be adequately hydrolyzed in the plasma and/or endothelial
cells to release NO.
[0102] In one embodiment, the present invention provides a method
of pairing a therapeutic agent with an appropriate nitric oxide
donor to create an effective prodrug molecule. The method
comprises: (i) obtaining in vitro or in silico pharmacokinetic or
pharmacodynamic data, (ii) placing the data into a
physiologically-based pharmacokinetic/pharmacodynamic model, and
(iii) generating output parameters from the
pharmacokinetic/pharmacodynamic model, wherein the output
parameters determine the pairing of a therapeutic agent with an
appropriate nitric oxide donor to create an effective prodrug
molecule.
[0103] In one embodiment, the pharmacokinetic model of the present
invention comprises (i) a seven compartment model which divides a
gastrointestinal tract into seven compartments, wherein said seven
compartment model describes gastrointestinal absorption of said
prodrug molecule; and (ii) a group of compartment models which
divides a body into plasma/blood and tissue compartments (such as
heart, kidney, and liver), wherein said group of compartment models
describes the time course of the therapeutic agent, the nitric
oxide donor, and nitric oxide in gastrointestinal tract, blood, and
tissues. Representative in vitro or in silico input data to the
model include pKa values, octanol/water partition coefficients,
solubility data, permeability values, metabolism data, hydrolysis
data, serum protein binding data, nitric oxide release rate,
pharmacokinetic and pharmacodynamic data of a therapeutic agent,
and stability data in gastric and intestinal environments.
[0104] The present invention also provides a prodrug molecule
selected by the above method, wherein the prodrug molecule
comprises a therapeutic agent and a nitric oxide donor. In general,
the therapeutic agent can be a nonsteroidal anti-inflammatory drug
or an antibiotic. Representative nonsteroidal anti-inflammatory
drugs include, but are not limited to, non-selective cyclooxygenase
isozyme inhibitors or cyclooxygenase-2 inhibitors. Examples of
non-selective cyclooxygenase isozyme inhibitor include
acetylsalicylic acid (CH.sub.3COOC.sub.6H.sub.4COOH), IBUPROFEN
(C.sub.13H.sub.18O.sub.2), NAPROXEN (C.sub.14H.sub.14O.sub.3,)
indomethacin (C.sub.19H.sub.16ClNO.sub.4), and diclofenac
(C.sub.14H.sub.10Cl.sub.2NNaO). Moreover, the cyclooxygenase-2
inhibitor may comprise a carboxyl group. An example of nitric oxide
donor is a diazeniumdiolate such as diazen-1-ium-1,2-diolate. And
one of ordinary skill in the art would readily apply an antibiotic
as a therapeutic agent in view of the teaching of the present
invention.
[0105] In another embodiment, there is provided a prodrug molecule
comprising a nonsteroidal anti-inflammatory drug and a nitric oxide
releasing moiety, wherein the moiety has a half-life that is longer
than the total time period for hydrolysis and absorption, and
wherein a therapeutic dosage of nitric oxide is released into
enterocytes, thereby protecting against damage caused by
gastrointestinal irritation, bleeding or ulceration. Moreover, a
therapeutic dosage of nitric oxide may be released into blood
stream, thereby protecting one or more organ system such as heart,
kidney, and cardiovascular system. In general, the therapeutic
agent can be a nonsteroidal anti-inflammatory drug or an
antibiotic, and an example of a nitric oxide releasing moiety is a
diazeniumdiolate such as diazen-1-ium-1,2-diolate.
[0106] The present invention also provides a prodrug molecule
comprising: (i) a nitric oxide releasing moiety linked to an amino
acid through a linkage that is susceptible to enzymatic hydrolysis
or cleavage, and (ii) a therapeutic agent directly linked to said
amino acid, or linked to said amino acid through a spacer, wherein
the linkage between the therapeutic agent and the spacer, or the
linkage between the spacer and the amino acid is susceptible to
enzymatic hydrolysis or cleavage, wherein the release of the nitric
oxide releasing moiety and the therapeutic agent from the prodrug
molecule can be controlled independently. In general, the linkage
susceptible to enzymatic hydrolysis or cleavage is an ester
linkage, thioester linkage, amide linkage, or sulfonamide linkage.
The amino acid in this prodrug molecule can be hydroxyproline,
glutamic acid, or aspartic acid. Furthermore, the amino acid may
also comprise a free or substituted amine or amine salt.
[0107] The present invention also provides a compound of the
formula I:
##STR00008##
wherein R.sup.1 is an uncarboxylated core of a non-steroidal
anti-inflammatory drug, (e.g. naproxen, aspirin, ibuprofen,
indomethacin, salicylic acid, mesalamine, flunixin, ketorolac,
tolfenamic acid, niflumic acid, mefenamic acid, meclofenamic acid,
flufenamic acid, enfenamic acid, etodolac, pirazolac, tolmetin,
bromofenac, fenbufen, mofezolac, diclofenac, pemedolac, sulindac,
suprofen, ketoprofen, tiaprofenic acid, fenoprofen, indoprofen,
carprofen, loxoprofen, ibuprofen, pranoprofen, bermoprofen,
zaltoprofen, flurbiprofen, tenoxicam, piroxicam, meloxicam,
lornoxicam, tenidap, paracetamol, salactamide); or a structure of
the formula II:
##STR00009##
wherein R.sup.8 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl.
[0108] X in the formula I can have a structure of the formula
III:
##STR00010##
wherein X.sup.2 is oxygen, sulfur, or NH and X.sup.3 is oxygen,
sulfur, or NH.
[0109] Alternatively, X.sup.1 in the formula I can have a structure
of the formula IV:
##STR00011##
wherein X.sup.4 is oxygen, sulfur, or NH and X.sup.5 is oxygen,
sulfur, or NH.
[0110] In another embodiment, X.sup.1 in the formula I can have a
structure of the formula V:
##STR00012##
[0111] In yet another embodiment, X.sup.1 in the formula I can have
a structure of the formula VI:
##STR00013##
where X.sup.6 is oxygen, sulfur, or NH.
[0112] R.sup.2 in the formula I can be hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl.
[0113] R.sup.3 in the formula I can be hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl.
[0114] R.sup.4 in the formula I can be hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl; or a structure of the formula VII:
##STR00014##
wherein R.sup.9 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl; an amide derivative linked via a carboxy group of an
amino acid e.g. .beta.-alanine, alanine, 2-aminobutyric acid,
6-aminocaproic acid, .alpha.-aminoisobutyric acid,
.alpha.-aminosuberic acid, arginine, asparagines, aspartic acid,
citrulline, .beta.-cyclohexylalanine, cysteine, 3,4-dehydroproline,
glutamic acid, glutamine, glycine, histadine, homocitrulline,
homoserine, hydroxyproline, .beta.-hydroxyvaline, isoleucine,
leucine, lysine, methionine, norleucine, norvaline, ornithine,
penicillamine, phenylalanine, phenylglycine, proline,
pyroglutamine, sarcosine, serine, statine, threonine, tryptophan,
tyrosine, valine, or an amide derivative of a polypeptide.
[0115] In another embodiment, R.sup.4 in the formula I is a
structure of the formula VIII:
##STR00015##
wherein X.sup.7 is oxygen, sulfur, or NH, and R.sup.10 is an
unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, an unsubstituted or substituted heteroaryl.
[0116] In yet another embodiment, R.sup.4 in the formula I is a
structure of the formula IX:
##STR00016##
wherein X.sup.8 is oxygen, sulfur, or NH; and R.sup.11 is a
hydrogen, an unsubstituted or substituted C.sub.1-12 straight chain
alkyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkyl, an unsubstituted or substituted C.sub.1-12 straight chain
alkenyl, an unsubstituted or substituted C.sub.3-12 branched chain
alkenyl, an unsubstituted or substituted benzyl, an unsubstituted
or substituted phenyl, an unsubstituted or substituted C.sub.1-4
aryl alkyl, an unsubstituted or substituted heteroaryl; and
R.sup.12 is a hydrogen, an unsubstituted or substituted C.sub.1-12
straight chain alkyl, an unsubstituted or substituted C.sub.3-12
branched chain alkyl, an unsubstituted or substituted C.sub.1-12
straight chain alkenyl, an unsubstituted or substituted C.sub.3-12
branched chain alkenyl, an unsubstituted or substituted benzyl, an
unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl.
[0117] R.sup.5 in the formula I can be hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl; a structure of formula VII, a structure
of formula VIII, or a structure of formula IX.
[0118] R.sup.6 in the formula I can be hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, a structure of formula VII, or a
structure of formula VIII.
[0119] R.sup.7 in the formula I can be hydrogen, an unsubstituted
or substituted C.sub.1-12 straight chain alkyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkyl, an unsubstituted or
substituted C.sub.1-12 straight chain alkenyl, an unsubstituted or
substituted C.sub.3-12 branched chain alkenyl, an unsubstituted or
substituted benzyl, an unsubstituted or substituted phenyl, an
unsubstituted or substituted C.sub.1-4 aryl alkyl, an unsubstituted
or substituted heteroaryl, a structure of formula VII, or a
structure of formula VIII, or NR.sup.6R.sup.7 is a cyclic
heterocycle of the formula X:
##STR00017##
[0120] wherein R.sup.13 is hydrogen, or a structure of formula
XI:
##STR00018##
wherein X.sup.9 is oxygen, sulfur, or NH, and R.sup.14 is hydrogen,
an unsubstituted or substituted C.sub.1-12 straight chain alkyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkyl, an
unsubstituted or substituted C.sub.1-12 straight chain alkenyl, an
unsubstituted or substituted C.sub.3-12 branched chain alkenyl, an
unsubstituted or substituted benzyl, an unsubstituted or
substituted phenyl, an unsubstituted or substituted C.sub.1-4 aryl
alkyl, an unsubstituted or substituted heteroaryl, an amino acid
wherein X.sup.9 is the amino group of the amino acid (e.g.
b-alanine, alanine, 2-aminobutyric acid, 6-aminocaproic acid,
a-aminoisobutyric acid, a-aminosuberic acid, arginine, asparagines,
aspartic acid, citrulline, b-cyclohexylalanine, cysteine,
3,4-dehydroproline, glutamic acid, glutamine, glycine, histadine,
homocitrulline, homoserine, hydroxyproline, b-hydroxyvaline,
isoleucine, leucine, lysine, methionine, norleucine, norvaline,
ornithine, penicillamine, phenylalanine, phenylglycine, proline,
pyroglutamine, sarcosine, serine, statine, threonine, tryptophan,
tyrosine, valine, or a polypeptide linked via an amino functional
group).
[0121] Alternatively, NR.sup.6R.sup.7 is a cyclic heterocycle of
the formula XII:
##STR00019##
wherein Y is a structure of the formula XIII:
##STR00020##
wherein Y is a structure of the formula XIV:
##STR00021##
wherein R.sup.15 is hydrogen, an unsubstituted or substituted
C.sub.1-12 straight chain alkyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkyl, an unsubstituted or substituted
C.sub.1-12 straight chain alkenyl, an unsubstituted or substituted
C.sub.3-12 branched chain alkenyl, an unsubstituted or substituted
benzyl, an unsubstituted or substituted phenyl, an unsubstituted or
substituted C.sub.1-4 aryl alkyl, an unsubstituted or substituted
heteroaryl.
[0122] The present invention also provides a compound of the
formula XV:
##STR00022##
wherein Z is a structure of the formula XIII, or a structure of the
formula XIV.
[0123] The present invention also provides a compound of the
formula XVI:
##STR00023##
[0124] The present invention also provides a compound of the
formula XVII
##STR00024##
where the sub structure of the formula XVII
##STR00025##
represents the core structure of the amino acids alanine,
2-aminobutyric acid, acid, .alpha.-aminosuberic acid, arginine,
asparagines, aspartic acid, citrulline, .beta.-cyclohexylalanine,
cysteine, 3,4-dehydroproline, glutamic acid, glutamine, glycine,
histadine, homocitrulline, homoserine, hydroxyproline,
.beta.-hydroxyvaline, isoleucine, leucine, lysine, methionine,
norleucine, novaline, ornithine, penicillamine, phenylalanine,
phenylglycine, proline, pyroglutamine, sarcosine, serine,
threonine, tryptophan, tyrosine, or valine; wherein R.sup.18 is a
structure of the formula XVIII
##STR00026##
[0125] Alternatively, R.sup.18 is a structure of the formula
XIX:
##STR00027##
or R.sup.18 is a structure of the formula XX:
##STR00028##
or R.sup.18 is a structure of the formula XXI:
##STR00029##
[0126] The present invention also provides for a structure of the
formula XXII:
##STR00030##
[0127] The present invention also provides a structure of the
formula XXIII:
##STR00031##
[0128] Compounds of the present invention which contain one or more
asymmetric atoms can exist and be used as optically pure
enantiomers, mixtures of enantiomers, mixtures of enantiomers of
pure diastereomers, mixtures of both enantiomers and diastereomers,
completely racemic mixtures. Compounds of the present invention
which contain one or more carbon-carbon double bonds may exist as
pure E or Z isomers or mixtures of these isomers. Compounds of the
invention which contain one or more carbon-nitrogen double bonds
may exist as pure E or Z isomers or mixtures of these isomers.
Compounds of the invention which contain one or more atropisomers
may contain pure isomers or mixtures of these isomers. The present
invention anticipates and includes all such isomers and mixtures
thereof.
[0129] Compounds of the present invention which contain at least
one functional group salifiable with acids (e.g. primary, secondary
or tertiary amines) can be transformed into the corresponding
salts. Organic acids which could be used in this capacity include
oxalic, tartaric, maleic, succinic, citric, trifluoroacetic acids.
Examples of inorganic acids which could be used in this capacity
are nitric, hydrochloric, sulfuric and phosphoric acids.
[0130] The invention being generally described, will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
EXAMPLE 1
Selecting Appropriate NO-NSAID Candidate
[0131] The main objectives of this example are to (1) provide in
vivo data (i.e. NSAID and NO kinetic data) to train the in silico
manifestation of the pharmacokinetic/pharmacodynamic model; (2)
validate model predictions; and (3) select appropriate NO-NSAID
candidate(s) for future development. An NO-NSAID candidate will be
declared as a lead when it shows a potential of maintaining its
original NSAID anti-inflammatory activity, backed up by NO
production and PK data, without its untoward gastrointestinal,
cardiovascular and kidney events.
[0132] Anti-inflammatory activities of non-selective and selective
NSAIDs were indirectly measured using biomarkers indicating their
ability to inhibit COX-1 and -2 activities. NO activities were
measured that were relevant to their potential ability to
counteract NSAID side effects such as cardiovascular and kidney
events.
[0133] Myocardial infarction and ischemic events in high risk
patients led one of the most popular COX-2 inhibitors to its demise
(Bresalier et al., 2005). Non-selective NSAIDS may also cause
similar problems due to their ability to inhibit COX-2. It has been
postulated that a shift in the ratio of PGI.sub.2 to thromboxane
A.sub.2 during NSAID treatment would provide early indications of
atherogenicity (Krotz et al., 2005). Adenosine diphosphate (ADP)
generation is an indicator of myocardial infarction which has no
connection with the arachidonic acid cascade (Borna et al., 2005).
ADP level has been shown to be lowered by NO. Long term NSAID use
has been linked to kidney damage and hypertension (Zafirovska et
al., 1993). After a single dose of NSAID, proximal tubular damage
has been demonstrated (Porter et al., 1999). This was associated
with an increase in the urine the ratio of alanine-amino-peptidase
(AAP) and creatinine.
[0134] Table 1 is a summary of the protocol for a study which was
conducted in male Wistar rats weighing 275-300 grams. The animals
were allowed to acclimatize for at least five days prior to the
commencement of the study. The study protocol was approved by the
local animal ethics committee.
TABLE-US-00001 TABLE 1 A Protocol for Evaluating NAPROXEN
Derivative Diazeniumdiolate NO-NSAID Candidates No. of Animals
Dosage Dosage Dosage per Level Concentration Volume Treatment Group
Treatment Group (mg/kg) (mg/mL) (mL/kg) VEH vehicle 5 NA NA 5 NAP
naproxen 5 250 50 5 PYRO/NO- pyrrolidino- 5 Molar Specified in 5
NAP diazeniumdiolate- equivalent raw data naproxen to the NAP group
DMA/NO- dimethylamino- 5 Molar Specified in 5 NAP diazeniumdiolate-
equivalent raw data naproxen to the NAP group AZD3582
AstraZeneca/Nicox- 5 Molar Specified in 5 naproxen nitrate
equivalent raw data ester 2 to the NAP group ROF Rofecoxib 10 5 250
50 5
[0135] The animals were fasted overnight prior to test substance
administration. The test substance was administered orally by
gavage on the morning of the study day. All test animals had blood
collected during the test period in both EDTA
(ethylenediaminetetraacetic acid) tubes and SST (Serum Separator
Tubes) at 1, 3 and 6 hours after dosing. Blood was collected into
EDTA tubes only at 1 and 3 hours by tail tip amputation. Volume of
blood collected was between 0.5 and 1 mL at each of these
collections. The final collection was by puncture of the abdominal
vena cava under isoflurane general anesthesia. Final blood
collection was targeted as follows: 1.sup.st EDTA #1--1.5 mL;
2.sup.nd SST--1.5 mL; 3.sup.rd EDTA #2--as much as possible.
[0136] EDTA samples were centrifuged, and the plasma portion was
collected and frozen at -80.degree. C. SST samples were incubated
at 37.degree. C. for approximately 45 minutes, centrifuged, and the
serum was collected and frozen at -80.degree. C. until
analysis.
[0137] All test animals, housed in metabolic cages, had urine
collected over wet ice for 6 hours after dosing. Aliquots of the
urine from each animal were collected to determine creatinine
concentration. The remainder of the urine was centrifuged at 1000 g
for 10 minutes. The supernatant was collected and mixed with
analytical grade ethylene glycol at a rate of 0.4 mL ethylene
glycol per 1 mL urine supernatant. The ethylene glycol/urine
mixture was stored at -80.degree. C. until analysis.
[0138] All rats were euthanized by removal of the heart 6 hours
after initial dosing, following final blood collections.
Immediately after terminal blood collections were complete, the
thorax was opened and the heart removed. The heart was cut in half
longitudinally. Half of the heart was placed in formalin for
histological examination. The other half was quickly rinsed in
saline and then freeze-clamped (crushed between the jaws of a pair
of modified tongs, cooled by liquid nitrogen). An entire kidney was
freeze-clamped. The other kidney was left in situ for examination
by the pathologist. Freeze-clamped tissues were wrapped in labeled
aluminum foil and stored in liquid nitrogen until they could be
transferred to a -80.degree. C. freezer. Freeze-clamped tissues
were shipped on dry ice to NOVOKIN for analysis.
[0139] Each animal from all groups underwent a full necropsy under
the supervision of a board certified veterinary pathologist. The
stomach of each rat was cut along the greater curvature, contents
removed into a polypropylene (Falcon) tube, the mucosa rinsed with
saline and any obvious ulcers or erosions were measured along the
longest axis. This measurement was recorded for each ulcer or
erosion observed in each stomach. The falcon tubes and their
contents were frozen at -80.degree. C. and shipped to Novokin on
dry ice for analysis.
[0140] The stomach, duodenum, jejunum, ileum, cecum, colon, liver,
kidneys and heart were examined and collected into 10% neutral
buffered formalin. Two stomachs from each group regarded as being
representative of that group had their mucosal surfaces
photographed. Other tissues were also photographed.
[0141] The above tissues were all processed for histopathological
examination using hematoxylin and eosin staining. Additional
unstained slides were provided to the sponsor for TUNEL
staining.
[0142] The aggregate length of all gastric ulcers found in a given
animal was calculated. The mean aggregate ulcer length across
animals in a group was calculated. This mean value was reported as
the ulcer index for that group.
[0143] The ulcer index of each modified drug was compared with the
ulcer index of its associated parent drug and the controls using
analysis of variance and Duncan's multiple range test for pairwise
comparisons. Statistical analysis was done using SAS.RTM. (SAS
Institute Inc., Cary, N.C.).
[0144] The results of this study showed that naproxen and AZD3582
were severely ulcerogenic (Table 2, FIG. 7). DMA/NO-NAP was
somewhat less ulcerogenic, while PYRO/NO-NAP and ROF were not
significantly different from vehicle.
[0145] AUC.sub.0-6h (.mu.M-h) values for NAPROXEN after dosing with
the prodrug candidates or AZD3582 (Table 3, FIG. 8) were much lower
than for NAPROXEN parent drug, indicating that all three compounds
had poorer absorption than the parent drug.
[0146] Release of NO as indicated by the serum nitrate
concentrations at 6 hours (Table 4, FIG. 9) was similar among the
NO donors but erratic, resulting mostly in trends toward higher
concentrations even though the means were several-fold higher than
ROF or VEH.
[0147] NAP lowered both PGI2 and TXA2 levels (Table 5, FIG. 10),
leading to an insignificant change in the PGI2/TXA2 ratio when
compared to control (p>0.05). Both diazeniumdiolate compounds
have higher ratios than NAP with DMA-NO-NAP ratios achieving
statistical significance (p<0.05), suggesting decreased
cardiovascular risk. In contrast, both ROF, in keeping with
literature reports, but also AZD3582 exhibited lower ratios than
vehicle, suggesting possible increased risk although this was only
a trend in this experiment. The cardiac ADP levels and all of the
other potential nucleotide levels and ratios indicating energetic
stress on the heart remained the same across all groups, indicating
that this biomarker is insensitive to any particular toxicity
exerted by this group of compounds (data not shown).
[0148] NAPROXEN and its diazeniumdiolate prodrug compounds caused
no significant increase in the urine AAP/creatinine ratio (Table 6,
FIG. 11) while ROF and AZD3582 did cause an increase in this
biomarker, indicating proximal tubular kidney damage by these two
compounds. None of the compounds caused a significant change in
NAG/cr (Table 7, FIG. 12), indicating a lack of toxicity toward
distal tubules.
[0149] In summary, the new NO-NSAID candidates appeared to be
effective and have the following advantages over ROFECOXIB and
AZD3582: (a) plasma nitrite and nitrate levels increased; (b) ulcer
indices were lower than that of NAPROXEN or AZD3582 but not
ROFECOXIB treated animals. The results were similar to that of the
control for the new compounds; (c) cardiac PGI2/TXB2 was higher
than vehicle for NAPROXEN and the new compounds, and lower for
ROFECOXIB and AZD3582 suggesting a cardiotoxicity benefit with
diazeniumdiolate NO-donors; (d) urine AAP/creatinine ratio was
similar to that of the control and lower than that of the AZD3582
and ROFECOXIB treated animals, suggesting a renal toxicity benefit
to the diazeniumdiolates.
[0150] These results were compared to those of the corresponding in
vitro microsomal data. Because the NO-NSAID candidate is estimated
to release NO completely in the gut and the in vivo data show that
the new compounds have cardiac and kidney effects, it suggests that
NO is being transported at least somewhat by carriers such as
nitrosothiols and NO-hemoglobin. This comparison provided
information that is of tremendous value in the future design of
NO-NSAID and input value of the pharmacokinetic/pharmacodynamic
model.
[0151] This example suggests that in spite of potential NO-related
benefits shown in each biomarker category, the actual
reproducibility and degree of improvement may not be sufficient to
ensure commercial success of these compounds. The reason is that
the dosage used in this study is in the toxic range and the amount
of NO release will be at least several times lower at clinical
doses. An in vitro study (Knaus et al., 2005) showed that the
diazeniumdiolate candidates used in this study have 13 times the
capacity of producing NO when compared to the candidates such as
AZD 3582 which contains organic nitrates as NO donors.
Interestingly, the nitrate levels observed in this study is nowhere
close; suggesting a lot of NO has been "wasted" in vivo (FIG. 9).
Therefore, it will be necessary to further improve the solubility,
permeability and NO release characteristics over the studied
compounds before taking a new compound through the development
process. In particular, a more stable or stabilized NO donor to
link to NAPROXEN would improve systemic delivery and hence cardiac
and renal distribution/NO exposure of these organs, increasing the
benefit derived from the NO. This example demonstrates the
necessity for improvements to the Knaus et al. molecules, and these
can effectively and efficiently be directed through the use of
pharmacokinetic/pharmacodynamic modeling conducted in silico.
TABLE-US-00002 TABLE 2 Results of Duncan's Multiple Range Test For
Pairwise Comparisons of Ulcer Indices Category Mean Groups VEH
0.000 A ROF 0.000 A B PYRO-NO-NAP 1.450 B C DMA-NO-NAP 1.695 C D
AZD 3582 3.444 D E NAP 4.282 E Pairs with the same letter are not
significantly different (p > 0.05).
TABLE-US-00003 TABLE 3 Results of Duncan's Multiple Range Test For
Pairwise Comparisons of AUC.sub.0-6h (.mu.M-h) Values Category Mean
Groups PYRO-NO-Nap 99.262 A DMA-NO-Nap 150.570 A B AZD 3582 173.168
B Naproxen 501.606 C Pairs with the same letter are not
significantly different (p > 0.05).
TABLE-US-00004 TABLE 4 Results of Duncan's Multiple Range Test For
Pairwise Comparisons of Serum Nitrate Concentrations Category Mean
Groups VEH 51.424 A NAP 55.167 A B ROF 55.818 A B C PYRO-NO-NAP
389.000 A B C AZD 3582 460.667 B C DMA-NO-NAP 768.000 C Pairs with
the same letter are not significantly different (p > 0.05).
TABLE-US-00005 TABLE 5 Results of Duncan's MULTIPLE RANGE TEST FOR
PAIRWISE COMPARISONS of Cardiac Tissue PGI2/TXB2 Category Mean
Groups ROF 2.765 A AZD 3582 3.432 A VEH 3.562 A B NAP 4.544 B C
PYRO-NO-NAP 4.937 C DMA-NO-NAP 5.157 C Pairs with the same letter
are not significantly different (p > 0.05).
TABLE-US-00006 TABLE 6 Results of Duncan's Multiple Range Test For
Pairwise Comparisons of AAP/Cr. Category Mean Groups VEH 100.000 A
DMA-NO-NAP 132.412 A B PYRO-NO-NAP 195.569 A B NAP 263.788 A B C
AZD 3582 403.400 B C ROF 538.367 C Pairs with the same letter are
not significantly different (p > 0.05).
TABLE-US-00007 TABLE 7 Results of Duncan's Multiple Range Test For
Pairwise Comparisons of Urine NAG/Cr values Category Mean Groups
VEH 16.193 A DMA-NO-NAP 20.403 A PYRO-NO-NAP 21.515 A ROF 24.176 A
AZD 3582 27.439 A NAP 30.865 A Pairs with the same letter are not
significantly different (p > 0.05).
EXAMPLE 2
Physiologically-Based Pharmacokinetic/Pharmacodynamic Model
[0152] The objective of this example is to demonstrate an
embodiment of an in silico physiologically-based pharmacokinetic
computer model which incorporates all of the principal processes
and parameters and which is able to generate output as
described.
[0153] The model consists of a number of compartments, each
representing a specific anatomic region. For each compound of
interest in the model, each compartment has a specific volume
(volume of distribution) and has a uniform interior concentration
("well-stirred" condition) of the compound (FIG. 13). Compounds are
transported between compartments with rates proportional to the
amount of material in the originating compartment (first-order
kinetics). These transports reflect diffusion and bulk flows
between physiologically adjoining compartments. Many of these
transports represent blood plasma circulation, and the sum of these
flows into any compartment is equal to the sum of all blood plasma
flows out of the compartment. Within a compartment, compounds
undergo metablokic reactions, producing metabolites in
stoichiometric proportions, at compound and compartment specific
rates. The new materials are produced in amounts proportional to
those of the original compounds (first-order kinetics)
[0154] The simulation consists of an arterial blood plasma
compartment (FIG. 26) with flows to an intestinal region (FIG. 14).
Flows lead from the intestinal region to a compartment representing
the liver (FIG. 25). The arterial blood plasma compartment also has
flows to four compartments representing heart (FIG. 23), liver
(FIG. 25), kidney (FIG. 24), and other tissues (FIG. 27),
respectively. Flows lead from these four compartments into a venous
blood plasma compartment. This has a flow into a lung compartment,
which in turn flows into the arterial blood plasma compartment. The
intestinal region is divided into seven segments, each comprised of
five compartments (FIGS. 15 to 21). One of the five compartments of
each intestinal segment represents the intestinal lumen and these
luminal compartments are connected in sequence to reproduce drug
transit including peristaltic behavior of the intestine. Within
each of the seven intestinal segments, the lumen compartment has
bi-directional flows with an enterocyte (absorptive cells lining
the lumen) compartment, which also has bi-directional flows with a
blood plasma compartment. A second blood plasma compartment has
bi-directional flows with the fifth compartment representing the
other intestinal tissues supplied by the cranial mesenteric artery.
Each blood plasma compartments receives an in-flow from the
arterial compartment has an equal output to the hepatic
compartment. No other intestinal compartments are connected to the
rest of the model. A final compartment with a flow into the first
lumen compartment is used to model an oral dose of drug (FIG. 15).
Physiological data from human and animals for body weight, cardiac
output, blood flow to various organs, volumes and weight of each
organ, extracellular and intracellular fluid, lipids, were
collected from the literature and are summarized in following
tables. In silico estimation of log P, plasma protein binding, and
per organ volumes of distribution, is accomplished using the
methods published by Ghose et al. (1998), Lobell and Sivarajah
(2003), and Poulin and Thiel, (2002), respectively.
[0155] Whenever in vitro estimate is attainable, the in vitro
results will be used, for example, plasma protein binding. Methods
reported by Bowalgaha & Miners (2001), Martignoni et al.
(2006), Tong et al. (2001) and Thulesen et al. (1999) were used for
in vitro and in vivo scale-up for clearance in the intestine,
absorption rate constant and hepatic clearance. The simulation
begins with no material in all compartments except for the initial
bolus in one compartment (typically the stomach compartment). The
simulation then estimates the changing distribution of the material
with time.
[0156] The current version of the simulation is implemented using
the MatLab and its Simulink Toolbox (both The Mathworks, Natick,
Mass.), and is a mixture of the Simulink graphical model interface,
MatLab command language, and a code-generation routines written in
Perl. The structure of the model is depicted in FIGS. 13-28.
EXAMPLE 3
Effects of NSAIDs and NO Donors on Platelet Aggregation
[0157] The objectives of this example are to: (1) study the effects
of NSAIDs and NO donors on platelet aggregation, vasodilation, and
thrombus formation; (2) study the potential interaction between
NSAIDs and NO donors in platelet aggregation, vasodilation and
thrombus formation; and (3) the effects of NSAIDs and NO on COX
functions.
[0158] The methods published by Al et al. (2006), Hanson et al.
(2005), Turkan et al. (2004) and Tubara et al. (2001) will be used
to achieve these objectives.
[0159] In vitro results obtained from these studies will be used to
simulate the time course of platelet aggregation, vasodilation and
thrombi formation after administration of NO-NSAID candidates.
EXAMPLE 4
Training of the Pharmacokinetic/Pharmacodynamic Model
[0160] The objectives of this example are: (1) to train the
physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD)
model and (2) to use the PBPK/PD model to predict in vivo
pharmacokinetic and pharmacodynamic behavior of potential
candidates in human and rat. The physiological, in vitro and in
silico inputs into the model are listed in Table 8.
[0161] NAPROXEN, AZD 3582 and PYOR/NO-NAPROXEN are used to train
the model. The in vitro parameters are generated in house unless
they are specified otherwise. The model parameters are listed in
Table 9.
TABLE-US-00008 TABLE 8 Input for the Physiologically-Based
Pharmacokinetic/Pharmacodynamic Model Parameter name Rat Human
Source Body mass, kg 0.25 70 Stomach Volume, mL 1.1 155 Bernareggi
and Rowland, 1991 Relative small intestine 1.40 0.91 Tables 5 &
7 - Brown et al., 1997 weight, % body weight Total lumen volume mL
2.0 Pang, 2003 Total plasma volume, enterocyte, 0.9 Pang, 2003 mL
Total plasma volume, serosa, mL 0.81 Pang, 2003 Intestinal
enterocyte/serosa ratio 1.11 * Pang, 2003 Intestinal radius, cm 0.2
2.0 RIVM report, 1999 Stomach to lumen clearance, 100 100
arbitrary, fast value mL/min Clearance to next lumen segment, 0.1
0.1 based 0.03 mL/min in Pang, 2002 mL/min Cardiac output, blood,
mL/min 110 5200 Table 22 - Brown et al., 1997 Plasma fraction of
blood 0.58 0.58 Table II - Davis, 1993 Fraction of aortic blood to
GIT 0.153 0.181 Table 23 - Brown et al., 1997 Fraction of aortic
blood to hepatic 0.02 0.046 '' artery Fraction of aortic blood to
kidneys 0.141 0.175 '' Fraction of aortic blood to heart 0.051 0.04
'' Fraction of intestinal blood to 0.3 * Pang, 2003 enterocytes *
Values reported for the rat were used in man.
TABLE-US-00009 TABLE 9 Input Parameters for the Simulation of AZD
3582, PYRO/NO-NAP (N-119) and NAPROXEN. Test Substances Rat Human
Source AZD3582 intestinal microsomal 0.09 554 In-house data
activity, mL/min/organ AZD3582 liver microsomal 1029 118566
In-house data activity, mL/min/organ AZD3582 intestinal
permeability, 30 .times. 7.0e.sup.-6 15 .times. 6.0e.sup.-6
Fagerholm et al., 2005 cm/s (human paper) AZD3582 to Naproxen,
intestinal 10 36 In-house data molar percent conversion, % AZD3582
to Naproxen, liver 40 72 In-house data molar percent conversion, %
AZD3582 gastric decay rate 0 0 In-house data constant, /mint
AZD3582 intestinal lumen decay 0 0 In-house data rate constant,
/min AZD3582 plasma decay rate 1.0 1.0 In-house data constant, /min
N-119 intestinal microsomal 1 188 In-house data activity,
mL/min/organ N-119 liver microsomal activity, 1920 567000 In-house
data mL/min/organ N-119 to Naproxen, intestinal 10 87 In-house data
molar percent conversion, % bN-119 to Naproxen, liver molar 40 72
In-house data percent conversion, % N-119 gastric decay rate
constant, 0 0 In-house data /mint N-119 intestinal lumen decay rate
0.025 0.025 In-house data constant, /min N-119 plasma decay rate
constant, 1.4 -- In-house data /min Naproxen intestinal 5e.sup.-5
8e.sup.-4 Fagerholm et al., 2005 permeability*, cm/s Naproxen liver
microsomal 0.0075 0.0092 Estimated from Table II- activity,
mL/min/organ Runkle, 1972 ** NAPROXEN has zero permeability from
intestinal enterocyte to lumen.
[0162] The output data are summarized in FIGS. 29-35 and Table 10.
In general, the PBPK/PD model adequately predicts the plasma
concentration of NAPROXEN (FIGS. 29 & 30) and NAPROXEN formed
from AZD 3582 (FIGS. 31 & 32 and 34 & 35) The predicted
values are within a two-fold range of the reported data. Consistent
with the data reported in the literature (Fagerholm et al., 2005
and Fagerholm & Bjornsson, 2005), the model also predicts
extremely low AZD 3582 bioavailability. The cause for the extensive
first-pass effect is due to its extensive hydrolysis in the
intestine and the liver. Both organs have significant contributions
to its first-pass removal in rat and human (FIGS. 33 and 36). Since
the fate of the metabolites which carry the NO donor is not known,
it is difficult to predict where NO is being generated. It should
be noted that rat and human are different in terms of first-pass
removal of AZD 3582. Liver first-pass is dominant in rat; whereas
intestinal first-pass is dominant in human. This example shows the
importance of using in vitro tests instead of using in silico
prediction when it comes to metabolism estimation. As a general
rule, the rate of metabolism in rat is faster than that in human;
however, we do see several differences in the present studies.
[0163] FIG. 35 shows the AUC of the diazeniumdiolate in the lumen,
gastrointestinal tissue, liver, heart, kidneys and the rest of the
body after oral N-119 administration. Since N-119 is not stable in
the intestinal lumen and the rate of hydrolysis is high in rat
intestinal microsomes (Table 9), this prodrug candidate released
most of its nitric oxide before it enters the liver. Is estimated
that systematic exposure of nitric oxide will be at a minimum. The
results of this simulation are consistent with that the relatively
low plasma nitrate level after the administration of
PYRO/NO-naproxen. The plasma nitrate level is not that much higher
than that of AZD 3582, although the NO generating capacity of
PYRO/NO-naproxen is 13 times higher than that of AZD 3582 in vitro
(Knaus et al., 2005). This set of data is consistent with the
speculation that the set of candidates generated by Knaus et al.
(2005) is not appropriate for further development, despite
observable effects in the heart and kidneys.
[0164] The pharmacokinetic behavior of AZD 3582 is similar to that
of PYRO/NO-NAPROXEN except that AZD 3582 is more stable in the
intestinal lumen and intestinal microsomes (Table 9). The release
profile of NAPROXEN (FIGS. 33-34), after the administration of AZD
3582, suggests that the nitric oxide donor was also released in the
first-pass organs. In the absence of measurable nitric oxide donor
and metabolite data, it is not possible to estimate when NO was
being generated. The in vivo study listed in Example 1 showed that
the in vivo plasma nitrate levels were not high enough to trigger
any observable effects in heart and kidneys (FIGS. 9-12). It is
deduced that there is not enough NO being generated in the systemic
circulation. This could be due to: (1) too much NO is being
generated in the intestinal lumen, intestine and liver; and/or (2)
the NO generating capacity is too low.
TABLE-US-00010 TABLE 10 Pharmacokinetic Parameters Estimated by the
Model Using in vitro and in silico Inputs Listed in Tables 8 and 9.
AZD3582 Naproxen Naproxen PK Parameters Rat Human Rat Human Dose,
mg 0.75 300 1.3 375 V.sub.d, L/kg 0.09 0.12 0.09 0.12 t.sub.1/2, hr
7.8 17.5 7.8 17.5 C.sub.max, .mu.M 50 125 55.2 83 T.sub.msa, hr
0.55 2.25 0.74 2.79
EXAMPLE 5
Developing Candidate Compounds
[0165] The present invention also provides a process of developing
and improving the pipeline of compounds such as the described
NO--NSAIDs using all of the elements described in the
aforementioned examples.
[0166] The process begins with several prototype compounds with
some of the desired characteristics, e.g. DMA/NO-NAPROXEN and
PYRO/NO-NAP based on the Knaus (2005) chemistry. The simulations
results of DMA/NO-NAPROXEN were similar to that of
PYRO/NO-NAPROXEN. For the sake of simplicity, the results of
PYRO/NO-NAPROXEN are shown above in Example 4.
[0167] The evaluations described in Example 4 show that the
pharmacokinetic model described in this invention is capable of
identifying imperfections of potential candidates. The candidates
designed by Knaus et al. (2005) (FIG. 36) have the advantage of
releasing a higher quantity of NO. However, these candidates lack
specificities in NO delivery in terms of targeting internal organs
such as heart and kidneys.
[0168] It becomes obvious from these simulations that an ideal
candidate should have an optimal log P value at physiological pH.
More importantly, the release of nitric oxide and NSAID should have
certain degree of specificity. For example, it would be desirable
to have a significant dose of NSAID released after prodrug
administration, such that the antiiflammatory action will take
effect soon. However, the NO donor should be less labile during the
first-pass after the prodrug administration.
[0169] Based on the simulation results, the present invention
describes a modular chemical library (FIG. 37) which can be used to
systematically control the physical properties and kinetics of a NO
prodrug so that systemic delivery of NO can be achieved. The goal
is to deliver an optimal dose of NO to the gastrointestinal tract,
heart and kidneys so that the potential side effects of NSAIDs can
be mitigated.
[0170] Examples of NO-NSAID prodrugs containing an amino acid have
been reported (Ranatunge et al., 2006; Kartasasmita et al., 2002;
Andersson et al., 2004; Gilmer et al., 2002; Almirante et al.,
2006; Benedini et al., 2000; Bolla et al., 2005; Rivolta et al.,
2005; Del Soldato, 2002a, 2002b, 2003, 2004a and 2004b). These are
limited to examples employing nitrooxyalkyl functionality as the
source of NO. The rate of NO release from these prodrugs (or
degradation products thereof) is determined by the rate of
nitrooxyalkyl reduction, which occurs via multiple pathways (Fung,
2004; Carini et al., 2002; Gao et al., 2005; Satyum, 2006) and is
therefore difficult to control.
[0171] In the present invention, the release rate of NO from the
prodrug can be systematically controlled. The mechanism of NO
release from the O.sup.2-substituted diazeniumdiolate occurs in two
distinct steps. Enzymatic release of the diazeniumdiolate from the
prodrug gives an O.sup.2-unsubstituted diazeniumdiolate which
subsequently undergoes rapid decomposition under physiological
conditions to release NO. If the correct O.sup.2-substituted
diazeniumdiolate is used, the NO-NSAID candidate will be stable
towards physiological pH. If the release rate of diazeniumdiolate
from the candidate compound via enzymatic hydrolysis
(t.sub.1/2=minutes to hours) exceeds the half life of the released
unsubstituted diazeniumdiolate to a sufficient degree (examples
include, but are not limited to, PROL-NO (t.sub.1/2 2 s, Keefer,
2005), PYRRO-NO (t.sub.1/2 3 s, Saavedra, 2000)), it can be
considered that enzymatic hydrolysis of the ester linkage between
the amino acid and diazeniumdiolate is the rate determining step
for NO release.
[0172] The rate of enzymatic release can be controlled by a number
of factors. In the NO-NSAID candidates developed by Knaus et al.
(2005) (FIG. 8), the O.sup.2-substituted diazeniumdiolate is
directly attached to the NSAID. Enzymatic cleavage results in
concomitant release of both the NSAID and O.sup.2-unsubstituted
diazeniumdiolate. The rate of enzymatic hydrolysis and therefore
the release rate of NO and NSAID is directly controlled by the
choice of the diazeniumdiolate. One limitation of this approach is
that in addition to altering the rate of enzymatic hydrolysis,
altering the diazeniumdiolate can affect both the half-life
(t.sub.1/2) and efficiency of NO generation. Furthermore, the
toxicity/metabolic profile will change because different secondary
amines will be generated during NO release. Although the list of
potential diazeniumdiolates derived from secondary amines and amino
acids is large, the number of biologically viable derivatives that
can be used in a prodrug is limited due to the toxicity of many
amines and/or their metabolic products (Mattioni et al., 2003;
Myers et al., 1997). It is therefore desirable to be able to
control the rate of release of a specific diazeniumdiolate
derivative with well characterized kinetic and toxicological
parameters when developing NO-NSAID candidates.
[0173] In the present invention, the NSAID or COX-2 inhibitor and
the O.sup.2-substituted diazeniumdiolate are independently attached
to a central amino acid via functionality (typically ester,
thioester, amide or sulfonamide) which is susceptible to enzymatic
hydrolysis or cleavage. This permits the enzymatic release of the
NSAID and diazeniumdiolate to occur at different rates. Control of
the absolute and relative release rates of NO (resulting from the
release of the diazeniumdiolate) and a specific NSAID can be
controlled by modifying the modules of the structure (FIG. 37),
specifically the amino acid, the amino acid nitrogen substituent,
and/or the spacer connecting the NSAID to the amino acid. Although
the choice of diazeniumdiolate will modify the rate of enzymatic
release, the enzymatic release rate can be changed by altering the
other modules while keeping the diazeniumdiolate constant. This
invention permits generation of new candidates with different
kinetic, physical, pharmacodynamic and pharmacokinetics properties
without the need to alter the NSAID or diazeniumdiolate.
[0174] Enzymatic degradation of the modular structure of the
present invention is designed to produce NO, a secondary amine,
formaldehyde, an N-substituted amino acid and the NSAID.
N-substituted amino acids can be considered as either prodrugs of
the corresponding parent amino acid, (Pitman, 1981) or additional
therapeutic agents (Chandran, 2005; Yu et al., 2006). Examples of
suitable N-substituents include (but are not limited to) amides
(Crankshaw et al., 2002), carbamates (Hansen et al., 1992) and
.alpha.-hydroxy or .alpha.-acyloxymethyls (Bundgaard et al.,
1987).
EXAMPLE 6
Controlling the Release Rate of Diazeniumdiolate from the NO-NSAID
Prodrug
[0175] Three examples of the modular library (1-3) were synthesized
based on three common modules; NAPROXEN as the NSAID,
hydroxyproline as the amino acid and DMA diazeniumdiolate. They
varied only in the nitrogen substituent of the amino acid, i.e.
free amine (1), acetyl (2) and pivaloyl (3) groups. Their chemical
stability was evaluated in phosphate buffer at different pH's over
a 30 minute period (Table 11). The free amine 1 underwent rapid
degradation over the pH range 2.5-7.0 to release the unsubstituted
diazeniumdiolate (not shown) and the NSAID-amino acid 4. The
N-acetyl derivative 2 was determined to undergo a much slower rate
of decomposition to generate the unsubstituted diazeniumdiolate
(not shown) and NSAID-amino acid 5 at pH 7.0. The prodrug 2 was
found to be stable at pH 2.5-5.0 over a 30 minute period. A
pivaloyl amide 3 was observed to be stable across the entire pH
range 2.5-7.0.
##STR00032##
TABLE-US-00011 TABLE 11 Loss of Diazeniumdiolate With Phosphate
Buffer at Various pH's Stability (phosphate buffer, 30 min)
Compound pH 2.5 pH 5.0 pH 7.0 1 x x x 2 x 3
[0176] The sensitivity of the prodrug 3 towards enzymatic
hydrolysis was evaluated by LC-MS. Liver and intestinal microsomal
preparations were used. It was found that cleavage of the
unsubstituted diazeniumdiolate resulting in the formation of the
NSAID-amino acid 6 was rapid in both the liver (complete hydrolysis
after 2 hours) and intestinal (50% conversion after 2 hours)
preparations. However, these rates were slower than the enzymatic
hydrolysis rates determined for the conversion of 1.fwdarw.4 and
2.fwdarw.5 (complete hydrolysis observed in 10 and 30 minutes
respectively in liver microsomal preparations).
##STR00033##
[0177] Enzymatic hydrolysis of the ester linking NAPROXEN to the
amino acid was found to be slow in all cases. After 2 hours, a 2%
release of naproxen was observed in liver microsomes.
[0178] A further embodiment of this invention is the recognition
that the use of two non-equivalent esters to independently release
the NSAID and diazeniumdiolate may proceed via the action of
specific esterases or other enzyme classes. As the distribution of
esterases varies throughout human tissues and organs, it is
possible for specific enzymes to release either the NSAID and/or
the diazeniumdiolate selectively at a specific target tissue or
organ.
[0179] A limitation of the NO-NSAIDS developed by Knaus et al.
(2005) is the exclusive use of NSAIDs that contain carboxylic acids
(FIG. 36). A similar limitation applies to the attachment of the
NSAID IBUPROFEN via an amide linkage to the distal nitrogen of a
piperazine diazeniumdiolate as reported by Saavendra et al. (1999).
A further embodiment of this invention is the ability to attach
other functionalized drugs or linker units (including but not
limited to alcohols, thiols, amines, sulfonamides) to the amino
acid module when the parent amino acid is a diacid such as aspartic
(n=1) or glutamic acid (n=2). This principle is demonstrated by the
diazeniumdiolate prodrugs of CELECOXIB and VALDECOXIB, potent and
selective COX-2 inhibitors (FIG. 38). In these examples, the drug
(CELECOXIB or VALDECOXIB) is attached to the amino acid of the
prodrug via its sulfonamide functionality to give structures 7 and
8 respectively.
[0180] A further embodiment of this invention is the recognition
that cleavage of either the NSAID/linker or the diazeniumdiolate
from a diacid amino acid module (including but not limited to
aspartic and glutamic acid) results in a pronounced reduction in
the rate of enzymatic hydrolysis of the remaining NSAID/linker or
diazeniumdiolate. This is exemplified by the di-NAPROXEN prodrug
(NAP-AA-NAP) 9 (FIG. 39). Initial enzymatic cleavage of both esters
occurs rapidly and non-selectively (complete hydrolysis after 30
minutes in liver microsomes) to give a mixture consisting
predominantly of NAPROXEN (AA-NAP) prodrugs 11 and 12. Cleavage of
the second linker released a further molecule of NAPROXEN from the
AA-NAP's 11 and 12. This however proceeds at a much slower rate
(40-65% conversion to 13 after 90 minutes).
[0181] Studies on di-DMA-diazeniumdiolate N-acyl glutamic acid
(DMA/NO-AA-DMA/NO) 14 (FIG. 40) have shown that an analogous change
of hydrolysis rate also occurs when esters link diazeniumdiolates
to glutamic and aspartic acid amino acids.
[0182] This principle can be further extended to a diazeniumdiolate
based NO-NSAID prodrugs (NO-AA-NSAID) 15 (FIG. 41). If the two
ester linkages for the NSAID and diazeniumdiolate moieties are
initially hydrolyzed at different rates (for example, the NSAID is
initially cleaved more rapidly than the diazeniumdiolate), the
NSAID will be predominantly released first from 15. This will
generate a mono acidic diazeniumdiolate prodrug ester (NO-AA) 16
and the free NSAID (or biologically relevant salts thereof). The
(NO-AA) 16 would subsequently undergo slower enzymatic release of
the diazeniumdiolate than is the case for the NO-AA-NSAID 15,
therefore providing a slow release of NO. conversely, if the
diazeniumdiolate was initially cleaved faster from the NO-AA-NSAID,
then there will be fast generation of diazeniumdiolate (and
therefore NO) with slow release of the NSAID
EXAMPLE 7
Differential Release of the NSAID and the Diazenium-Diolate from
NO-AA-NSAID
[0183] This principle of differential hydrolysis rates has been
demonstrated for NO-AA-NSAIDS CMD113 and CMD114 (FIGS. 42-43).
Exposure of both CMD113 and CMD114 to various enzymatic
preparations (FIGS. 44-51) resulted in rapid initial loss of the
prodrug candidate to give NAPROXEN and the NO-AA (FIG. 42) and
slower competitive release of the dimethylamino diazeniumdiolate to
give the AA-NAP (FIG. 43). Subsequent enzymatic hydrolysis of the
remaining ester in both the NO-AA (FIG. 42) and AA-NAP (FIG. 43)
proceeded at a much slower rate than the initial hydrolysis to give
N-pivaloyl glutamic acid.
[0184] It is important to note that the NO-AAs formed from CMD 113
and CMD 114 have several features in common. The first one is that
both compounds are relatively stable in both intestinal and liver
microsomes of human and rats (FIGS. 44-51). The second one is that
both of these compounds hydrolyzed in the rat plasma at a
relatively fast rate (Tables 12 & 13). It is clear that NO-AAs,
with similar structures, may be stable after they are released from
their respective prodrug moiety in intestinal and liver; but these
species will be able to release NOD and subsequently NO in plasma.
The difference in the response to intestinal and liver microsomes
vs. plasma is an important feature in the design of NO-AA. Ideally,
the structure of a potential NO-AA should be susceptible to all
esterases, with optimal release rates.
TABLE-US-00012 TABLE 12 Input Parameters for the Simulation of CMD
113 CMD 113 Rat Human Source Intestinal microsomal activity, 2 4813
In-house data mL/min/organ Liver microsomal activity, 6545 434763
In-house data mL/min/organ Intestinal molar conversion to 62 75
In-house data naproxen, % Liver molar conversion to 90 72 In-house
data naproxen, % gastric decay rate constant, /mint 0 0 In-house
data intestinal lumen decay rate 0.2 0.2 In-house data
constant,/min plasma decay rate constant, /min 1.2 -- In-house data
NO-AA half-life in plasma, min. 9.2 -- In-house data
TABLE-US-00013 TABLE 13 Input Parameters for the Simulation of CMD
114 CMD 114 Rat Human Source Intestinal microsomal activity, 9 5006
In-house data mL/min/organ Liver microsomal activity, 7667 721224
In-house data mL/min/organ Intestinal molar conversion to 42 78
In-house data naproxen, % Liver molar conversion to 60 50 In-house
data naproxen, % gastric decay rate constant, /mint 0 0 In-house
data intestinal lumen decay rate 0.67 0.67 In-house data
constant,/min plasma decay rate constant, /min -- -- In-house data
NO-AA half-life in plasma, min. 3.8 -- In-house data
[0185] This principle can be extended to include COX-2 inhibitors
(including but not limited to ROFECOXIB). Prodrugs of COX-2
inhibitors such as ROFECOXIB and some COX-2-inhibitors contain a
carboxylic acid or alcoholic functional handles (for examples see
Black et al., 1997, 1998a, 1998b, 1999), which can be used to
attach the molecule to the modular scaffold described herein. In
such cases, the COX-2 inhibitor prodrug (such as that shown in 18
based on a known ROFECOXIB prodrug 20 (Engelhardt et al., 2006)
will be released rapidly (as described previously for the analogous
NSAID derivatives), resulting in the same NO-AA 16 (FIG. 52). As
before, hydrolysis of the NO-AA 16 to release the diazeniumdiolate
21 (and therefore NO) will be significantly slower than the initial
hydrolysis step. This will provide for rapid release of the COX-2
inhibitor (or prodrug thereof) and slow release of the diazenium
diolate (and therefore NO).
EXAMPLE 8
General Methods for the Preparation of NONOate-Amino Acid-NAPROXEN
Prodrugs 26
N-Boc Protected NO-AA's 24 (FIG. 53)
[0186] A solution of the chloride 23 [(11.0 mmol), cf. Knaus et al.
(2005)], in hexamethylphosphorus triamide (HMPA) (5 mL) was added
to a suspension of the N-Boc amino acid 22 (9.13 mmol), (Engelhardt
et al., 2006) and Na.sub.2CO.sub.3 (9.13 mmol) in HMPA (5 mL) at
room temperature (rt) and the resulting mixture was stirred
overnight. Water was then added to the mixture and the resulting
aqueous layer was extracted with ethyl acetate (EtOAc) (.times.5).
The organic fractions were collected, dried (Na.sub.2SO.sub.4 or
MgSO.sub.4) and concentrated in vacuo. The residue was purified by
flash chromatography (silica gel) eluting typically with
hexane/EtOAc to give 24.
N-Boc Protected NO-AA-NAP's 25 (FIG. 53)
[0187] A solution of 24 (1.0 mmol), naproxen (1 mmol)
dicyclohexylcarbodiimde (DCC) (1.0 mmol) and
4-(dimethylamino)pyridine (DMAP) (0.1 mmol) in anhydrous
CH.sub.2Cl.sub.2 (10 mL) was stirred at rt for a period of 1
h--overnight [(the reaction was monitored by thin layer
chromatography (TLC)]. The resulting white precipitate was removed
by filtration and the filtrate was concentrated in vacuo. The
residue was purified by flash chromatography (silica gel) eluting
typically with hexane/EtOAc to give 25.
N-Acetyl NO-AA-NAP's (R.sup.5=Me) 26 (FIG. 53)
[0188] 25 (0.1 mmol) was dissolved in trifluoroacetic acid (TFA) (1
mL) at rt and stirred for 1-6 h (reaction monitored by TLC). The
resulting mixture was concentrated in vacuo. The residue was taken
up into acetic acid (AcOH) (1 mL) and acetic anhydride (Ac.sub.2O)
(0.18 mL) was added dropwise with stirring at rt. The resulting
mixture was stirred at rt overnight. The reaction mixture was then
concentrated in vacuo and the residue was purified by flash
chromatography (silica gel) eluting typically with EtOAc/CHCl.sub.3
to give 26.
N-Pivaloyl NO-AA-NAP's (R.sup.5.dbd.CMe.sub.3) 26 (FIG. 53)
[0189] 25 (1 mmol) was dissolved in TFA (5 mL) at rt and stirred
for 1-6 h (reaction monitored by TLC). The resulting mixture was
concentrated in vacuo. The residue was taken up into
CH.sub.2Cl.sub.2 (5 mL) and pivaloyl chloride (PivCl) (0.17 mL)
followed by Et.sub.3N (0.32 mL) were added dropwise with stirring
at rt. The resulting mixture was stirred at rt overnight. The
reaction mixture was then concentrated in vacuo and the residue was
purified by flash chromatography (silica gel) eluting typically
with EtOAc/hexane to give 26.
EXAMPLE 9
General Methods for the Preparation of N-Ac NAP-Glu-NAP 9
[0190] The method is shown in FIG. 54. A suspension of N-Ac-L-Glu
(250 mg, 1.3 mmol), chloride 27 [(362 mg, 1.3 mmol) Phelan et al.
(1989)], KI (50 mg, 0.3 mmol) and Na.sub.2CO.sub.3 (140 mg, 1.3
mmol) in anhydrous dimethylformamide (DMF) (10 mL) was stirred at
rt overnight. The reaction mixture was then concentrated in vacuo
and the resulting residue was then taken up into water. The pH was
adjusted to 2 using 1N HCl and the aqueous layer was then extracted
with EtOAc (.times.3). The organic fractions were collected, dried
(Na.sub.2SO.sub.4) and concentrated in vacuo. The residue eluting
initially with 1:1 hexane:EtOAc and then 2:1 EtOAc:hexane to give 9
as a white solid (132 mg, 30%).
EXAMPLE 10
General Methods for the Preparation of N-Ac DMA/NO-Glu-NO/DMA
14
[0191] The method is shown in FIG. 55. A suspension of the DMA
chloride 23 [(3.2 mmol), Knaus et al. (2005)], N-acetyl-L-glu (250
mg, 1.32 mmol) and Na.sub.2CO.sub.3 (280 mg, 2.64 mmol) in HMPA (3
mL) was stirred at rt overnight. Water was then added to the
mixture and the resulting aqueous layer was extracted with EtOAc
(.times.3). The organic fractions were collected, dried
(Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was
purified by flash chromatography (silica gel) eluting with EtOAc to
give 14 (239 mg, 43%).
EXAMPLE 11
General Methods for the Preparation of CMD113 and CMD114
N-Boc-O-Benzyl Glu-NAP 28 and 29
[0192] A mixture of the Boc-L-Glu benzyl ester (1.36 mmol),
chloride 27 [(1.36 mmol) Phelan et al. (1989)], KI (100 mg, 0.6
mmol) and Na.sub.2CO.sub.3 (150 mg, 1.36 mmol) in anhydrous DMF (10
mL) was stirred at rt overnight. The reaction mixture was then
concentrated in vacuo and the resulting residue was then taken up
into water. The aqueous layer was then extracted with EtOAc
(.times.3). The organic fractions were collected, dried
(Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was
purified by flash chromatography (silica gel) eluting typically
with EtOAc/hexanes to give the title compounds.
Deprotection of 1-Boc-O-Benzyl Glu-NAP 30 and 31
[0193] A suspension of the N-Boc-O-Benzyl Glu-NAP (0.79 mmol) and
5% Pd/C (50 mg) in EtOAc (50 mL) was stirred vigorously under an
atmosphere of H.sub.2 (1 atm) at rt 3-6 h (the reaction was
monitored) by TLC). The mixture was then filtered through a pad of
Celite and the filtrate was concentrated in vacuo to give the title
compounds.
Synthesis of N-Boc Protected DMA/NO-Glu-NAP 32 and 33
[0194] A suspension of the chloride 23 [(0.73 mmol), Knaus et al.
(2005)], N-Boc-O-Benzyl Glu-NAP (0.49 mmol) and Na.sub.2CO.sub.3
(78 mg, 0.74 mmol) in HMPA (5 mL) was stirred at rt overnight.
Water was then added to the mixture and the resulting aqueous layer
was extracted with EtOAc (.times.3). The organic fractions were
collected, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The
residue was purified by flash chromatography (silica gel) typically
eluting with EtOAc/hexane to give the title compounds.
N-Pivaloyl Glutamic Acid-NONOates CMD113 and CMD114
[0195] N-Boc Protected DMA/NO-Glu-NAP (0.31 mmol) was dissolved in
TFA (3 mL) at rt and stirred for 30 min-2 h (reaction monitored by
TLC). The resulting mixture was concentrated in vacuo. The residue
was taken up into CH.sub.2Cl.sub.2 (5 mL) and pivaloyl chloride (58
.mu.L) followed by Et.sub.3N (100 .mu.L) were added dropwise with
stirring at rt. The resulting mixture was stirred at rt overnight.
The reaction mixture was then concentrated in vacuo and the residue
was purified by flash chromatography (silica gel) eluting typically
with EtOAc/hexane to give the title compounds.
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